In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
This report summarizes an experiment identifying cardiac genes in embryoid bodies derived from embryonic stem cells. RNA was extracted from embryoid bodies at day 5 and day 8 after differentiation. cDNA was synthesized and PCR was performed using primers for cardiac, stem cell, and housekeeping genes. Gel electrophoresis showed expression of cardiac genes α-mhc, gata4, and cxn40 as well as the housekeeping gene gapdh in both samples. Real-time PCR results were inconclusive due to unexpected amplification curves. The experiment provides evidence that embryoid bodies can differentiate into cardiomyocytes expressing characteristic cardiac genes.
Identification of perfect housekeeping genes for gene expression studies in p...ICRISAT
Gene expression analysis using quantitative real-time PCR (qRTPCR) is a very sensitive technique which completely depends on stable performance of reference genes used in the study.
3 July 2014
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
The document summarizes research on the Monascus genome project and related genetic studies. Key points include:
1) The Monascus genome was sequenced to 8x coverage, revealing various genomic features. A Monacolin K biosynthesis gene cluster was identified containing genes for polyketide synthases and other enzymes.
2) An efficient method for genetic transformation of Monascus pilosus was developed using aurintricarboxylic acid.
3) Disruption of the mokA gene and overexpression of the mokH transcription factor gene resulted in loss of and increased Monacolin K production, respectively.
PRODUCTION OF SEROTYPE 6-DERIVED RECOMBINANT ADENO-ASSOCIATED VIRUS IN SERUM-...Dr. Érica Schulze
This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Stable 16 year storage of DNA purified with the QIAamp® DNA Blood mini kit - ...QIAGEN
In this application note, we describe the success of the QIAamp DNA Blood Mini Kit in the preparation of highly stable DNA,as evidenced by 16-year storage data. We also report the best storage conditions for maximal protection against degradation.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
This report summarizes an experiment identifying cardiac genes in embryoid bodies derived from embryonic stem cells. RNA was extracted from embryoid bodies at day 5 and day 8 after differentiation. cDNA was synthesized and PCR was performed using primers for cardiac, stem cell, and housekeeping genes. Gel electrophoresis showed expression of cardiac genes α-mhc, gata4, and cxn40 as well as the housekeeping gene gapdh in both samples. Real-time PCR results were inconclusive due to unexpected amplification curves. The experiment provides evidence that embryoid bodies can differentiate into cardiomyocytes expressing characteristic cardiac genes.
Identification of perfect housekeeping genes for gene expression studies in p...ICRISAT
Gene expression analysis using quantitative real-time PCR (qRTPCR) is a very sensitive technique which completely depends on stable performance of reference genes used in the study.
3 July 2014
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
The document summarizes research on the Monascus genome project and related genetic studies. Key points include:
1) The Monascus genome was sequenced to 8x coverage, revealing various genomic features. A Monacolin K biosynthesis gene cluster was identified containing genes for polyketide synthases and other enzymes.
2) An efficient method for genetic transformation of Monascus pilosus was developed using aurintricarboxylic acid.
3) Disruption of the mokA gene and overexpression of the mokH transcription factor gene resulted in loss of and increased Monacolin K production, respectively.
PRODUCTION OF SEROTYPE 6-DERIVED RECOMBINANT ADENO-ASSOCIATED VIRUS IN SERUM-...Dr. Érica Schulze
This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Stable 16 year storage of DNA purified with the QIAamp® DNA Blood mini kit - ...QIAGEN
In this application note, we describe the success of the QIAamp DNA Blood Mini Kit in the preparation of highly stable DNA,as evidenced by 16-year storage data. We also report the best storage conditions for maximal protection against degradation.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This study analyzed the expression of polysaccharide lyase (PL) sequences in soybean plants infected with the pathogen Phytophthora sojae. RNA was extracted from infected soybean leaves at 12, 24, 36, and 48 hours post infection. Reverse transcription PCR was used to obtain cDNA from the RNA samples. Quantitative PCR analysis found that PL sequence expression was higher at 12 hours post infection than 24 hours for sequences that yielded usable data. This indicates that PL sequences are downregulated from 12 to 24 hours during P. sojae infection, though still upregulated compared to expression in P. sojae mycelium grown in vitro. The goal was to better understand the role of these enzymes in P.
Evaluation of Automated COBAS AMPLICOR PCR System for Detection of Several In...Alberto Cuadrado
We evaluated the COBAS AMPLICOR (CA) PCR system (Roche Diagnostic Systems) designed for automated
PCR amplification and detection of nucleic acids from infectious agents in clinical samples. The Roche
AMPLICOR microwell plate (MWP) PCR was the reference method. CA amplifies target nucleic acid, captures
the biotinylated amplification products by using magnetic particles coated with specific oligonucleotide probes,
and detects the bound products colorimetrically. For Mycobacterium tuberculosis, the correlation of the results
of CA tests with those of MWP tests was 100% with 230 samples, including 20 culture-positive samples. For
hepatitis C virus, the correlation was 100% with 214 samples, including 60 positive samples. MultiPlex CA
analysis of 199 cervical specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and the internal control
gave 100% concordance. These samples included 19 C. trachomatis and 3 N. gonorrhoeae culture-positive
samples. Overall, the agreement between PCR methods for all 842 comparisons was 100%. Compared with
culture, the sensitivities of the assays for C. trachomatis and M. tuberculosis were >95%. After spiking alternating
amplification tubes in the CA system with 1014 copies of the Chlamydia amplicon per ml, we were unable
to demonstrate any carryover cross-contamination of negative samples. Using the criteria of the College of
American Pathologists workload recording method, we found that the total hands-on time to produce CA PCR
results was 4.4, 7.9, and 3.3 min for M. tuberculosis, hepatis C virus, and the MultiPlexed assay for chlamydia
plus gonorrhea and an internal control, respectively. The CA system brings true PCR automation to laboratories.
In addition to the accuracy of automated results, the CA system provides labor savings, provides
containment of the amplification and detection components of PCR, and supports both MultiPlex amplification
and sequential algorithm (ReFlex) detection of analytes
Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...Thermo Fisher Scientific
The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
Automated DNA purification from diverse Microbiome samples using dedicated Mi...QIAGEN
This application note demonstrates the automation of QIAGEN’s new line of DNA sample prep kits for the microbiome. The microbiome of samples as diverse as soil, water and stool was purified using dedicated QIAcube compatible kits. Automation on the QIAcube enabled efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, the CLC Microbial Genomics Module was successfully employed for metagenome sequencing and identification of microbial composition and diversity.
This document outlines the development of a pipeline to characterize novel small RNAs discovered in clinical blood samples via next generation sequencing. Eight novel sequences were chosen as a pilot set. The pipeline involves verifying the sequences with qPCR, cloning and sequencing the products, and performing further analysis including transfection with inhibitors to determine functional significance. Two sequences, Cad 3 and Cad 7, were analyzed in more depth through the initial steps to help establish the pipeline. The goal is to ultimately understand the functional roles of these novel small RNAs.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
This document summarizes research on the effects of temperature on longevity in C. elegans. Microarray analysis identified lysosomal protease genes that are differentially expressed at 15°C vs 25°C. Follow up experiments showed that lysosome structure and number are altered at elevated temperatures. RNAi of temperature-regulated proteases caused progressive paralysis specifically at 15°C, indicating a role in neuronal health. Further experiments traced this paralysis to dysfunction of motor neurons rather than muscles. The research suggests that proteostasis, particularly lysosomal function, is important for neuronal health under cool temperatures and influences longevity.
1) The document describes an automated process for in vitro selection that was developed to generate nucleic acid aptamers faster than the traditional manual selection process.
2) An augmented Beckman Biomek 2000 pipetting robot was programmed to automate the major steps of in vitro selection, including preparing and purifying RNA, filtering RNA-target complexes, and amplifying selected sequences, in order to reduce the time needed to select aptamers from weeks or months to just days.
3) Initial attempts at automated selection yielded replication parasites but optimization suppressed their emergence and enabled the selection of true nucleic acid ligands binding to targets.
This document summarizes work done on culturing crocodile cell lines and cloning the parc gene from Pseudomonas keratitis. Primary crocodile cell lines were established from various organs and immortalized using hTERT. The parc gene was cloned from mutant and wild-type Pseudomonas strains and will be expressed and crystallized to study its role in quinolone antibiotic resistance.
Sequencing the transcriptome reveals complex layers of regulation, Department...Copenhagenomics
This document summarizes a study that analyzed gene expression and regulation in adipose tissue from obese and non-obese individuals. MicroRNA expression was found to be different between the two groups, with many miRNAs downregulated in obesity. One miRNA in particular, miR-193b, was shown to regulate secretion of the inflammatory factor CCL2. Motif activity response analysis identified transcription factors with significantly different activity between obese and non-obese individuals. Together, the results provide new insights into the perturbed transcriptional regulation of adipogenesis and inflammation in human obesity.
This study aimed to clone homologous genes of SND1, a key regulator of secondary cell wall biosynthesis, from Populus trichocarpa. The researcher amplified four SND1 homologs from P. trichocarpa cDNA using PCR. The amplified genes were cloned into E. coli and sequenced. Two colonies were found to contain the correct SND1 sequence insert, while others were false positives. Further work will express and quantify the proteins and determine their effects on other genes and phenotypes.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
The document discusses various molecular cytogenetics techniques including polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and fluorescent in situ hybridization (FISH). It provides details on the principles, techniques, and applications of PCR and RT-PCR. PCR is described as a technique that amplifies specific DNA regions, allowing minute quantities to be analyzed. Key steps involve DNA denaturation, primer annealing, and fragment extension. RT-PCR involves first converting RNA to cDNA then amplifying a specific region. Both techniques have numerous diagnostic and research applications.
olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
This document describes a real-time PCR array for simultaneously evaluating the expression of multiple cytokine mRNAs. The RT2Profiler PCR Array demonstrated high reproducibility, specificity, efficiency, sensitivity, and linear dynamic range. Using this array, 29 genes were found to have at least a 5-fold change in expression between resting and stimulated peripheral blood mononuclear cells after 6 hours of stimulation. The array provides a reliable tool for profiling cytokine pathway gene expression.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The discovery of circulating tumor DNA (ctDNA) in blood, urine
and other bodily fluids has led to a new type of non-invasive
method of characterizing cancer-causing mutations, the liquid
biopsy. With NGS technologies becoming increasingly
sensitive, down to a Limit of Detection (LOD) of 0.1%, they are
rapidly gaining traction as a valid assay for cancer genotyping
and have potential to direct cancer treatment plans. The wideangle
view provided by NGS panels, combined with digital
PCR’s zoomed-in precision detection of DNA provide a
comprehensive picture of a cancer’s genetic makeup. By
applying these complementary techniques at the appropriate
time based on the disease type and stage, cancer treatment
becomes quicker, more precise and more cost-effective in the
future. NGS and digital PCR (dPCR) together provide a
complete picture of the cancer genome.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
A novel method for building custom ampli seq panels using optimized pcr primers Thermo Fisher Scientific
AmpliSeq™ is a next generation sequencing library preparation method for targeted re-sequencing that utilizes highly multiplexed PCR to amplify regions of interest. A key to successful AmpliSeq libraries is the primer panel used for target amplification. Until now primers have been available as pre-assembled ready-to-use panels, or as custom made-to-order panels. We describe a new process for creating customized panels consisting of optimized and verified PCR primers. The primer sets are available as whole genes (i.e., all of the primers needed to create libraries that cover the entire coding regions of genes) and are selectable on the ampliseq.com website by either uploading gene lists or choosing genes from disease research areas.
We show NGS sequencing data from 10 disease research-oriented panels, including newborn screening research and inherited cancer research, assembled from individual pre-verified gene sets. Panel performance data include coverage uniformity, reproducibility, and sensitivity and positive predictive value of variant calling. To demonstrate flexibility of panel content and performance, the coverage uniformity of the 59 genes recommended by the American College of Medical Genetics and Genomics for reporting of incidental findings (ACMG59) was evaluated by themselves and with up to 135 additional genes and shown to be ≥ 97% in all contexts. We also demonstrate the robustness of this method using a variety of sample types (fresh, frozen, and dried blood, cheek swabs) with both manual and fully automated library preparation methods. For Research use only. Not for use in diagnostic procedures.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This study analyzed the expression of polysaccharide lyase (PL) sequences in soybean plants infected with the pathogen Phytophthora sojae. RNA was extracted from infected soybean leaves at 12, 24, 36, and 48 hours post infection. Reverse transcription PCR was used to obtain cDNA from the RNA samples. Quantitative PCR analysis found that PL sequence expression was higher at 12 hours post infection than 24 hours for sequences that yielded usable data. This indicates that PL sequences are downregulated from 12 to 24 hours during P. sojae infection, though still upregulated compared to expression in P. sojae mycelium grown in vitro. The goal was to better understand the role of these enzymes in P.
Evaluation of Automated COBAS AMPLICOR PCR System for Detection of Several In...Alberto Cuadrado
We evaluated the COBAS AMPLICOR (CA) PCR system (Roche Diagnostic Systems) designed for automated
PCR amplification and detection of nucleic acids from infectious agents in clinical samples. The Roche
AMPLICOR microwell plate (MWP) PCR was the reference method. CA amplifies target nucleic acid, captures
the biotinylated amplification products by using magnetic particles coated with specific oligonucleotide probes,
and detects the bound products colorimetrically. For Mycobacterium tuberculosis, the correlation of the results
of CA tests with those of MWP tests was 100% with 230 samples, including 20 culture-positive samples. For
hepatitis C virus, the correlation was 100% with 214 samples, including 60 positive samples. MultiPlex CA
analysis of 199 cervical specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and the internal control
gave 100% concordance. These samples included 19 C. trachomatis and 3 N. gonorrhoeae culture-positive
samples. Overall, the agreement between PCR methods for all 842 comparisons was 100%. Compared with
culture, the sensitivities of the assays for C. trachomatis and M. tuberculosis were >95%. After spiking alternating
amplification tubes in the CA system with 1014 copies of the Chlamydia amplicon per ml, we were unable
to demonstrate any carryover cross-contamination of negative samples. Using the criteria of the College of
American Pathologists workload recording method, we found that the total hands-on time to produce CA PCR
results was 4.4, 7.9, and 3.3 min for M. tuberculosis, hepatis C virus, and the MultiPlexed assay for chlamydia
plus gonorrhea and an internal control, respectively. The CA system brings true PCR automation to laboratories.
In addition to the accuracy of automated results, the CA system provides labor savings, provides
containment of the amplification and detection components of PCR, and supports both MultiPlex amplification
and sequential algorithm (ReFlex) detection of analytes
Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...Thermo Fisher Scientific
The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
Automated DNA purification from diverse Microbiome samples using dedicated Mi...QIAGEN
This application note demonstrates the automation of QIAGEN’s new line of DNA sample prep kits for the microbiome. The microbiome of samples as diverse as soil, water and stool was purified using dedicated QIAcube compatible kits. Automation on the QIAcube enabled efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, the CLC Microbial Genomics Module was successfully employed for metagenome sequencing and identification of microbial composition and diversity.
This document outlines the development of a pipeline to characterize novel small RNAs discovered in clinical blood samples via next generation sequencing. Eight novel sequences were chosen as a pilot set. The pipeline involves verifying the sequences with qPCR, cloning and sequencing the products, and performing further analysis including transfection with inhibitors to determine functional significance. Two sequences, Cad 3 and Cad 7, were analyzed in more depth through the initial steps to help establish the pipeline. The goal is to ultimately understand the functional roles of these novel small RNAs.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
This document summarizes research on the effects of temperature on longevity in C. elegans. Microarray analysis identified lysosomal protease genes that are differentially expressed at 15°C vs 25°C. Follow up experiments showed that lysosome structure and number are altered at elevated temperatures. RNAi of temperature-regulated proteases caused progressive paralysis specifically at 15°C, indicating a role in neuronal health. Further experiments traced this paralysis to dysfunction of motor neurons rather than muscles. The research suggests that proteostasis, particularly lysosomal function, is important for neuronal health under cool temperatures and influences longevity.
1) The document describes an automated process for in vitro selection that was developed to generate nucleic acid aptamers faster than the traditional manual selection process.
2) An augmented Beckman Biomek 2000 pipetting robot was programmed to automate the major steps of in vitro selection, including preparing and purifying RNA, filtering RNA-target complexes, and amplifying selected sequences, in order to reduce the time needed to select aptamers from weeks or months to just days.
3) Initial attempts at automated selection yielded replication parasites but optimization suppressed their emergence and enabled the selection of true nucleic acid ligands binding to targets.
This document summarizes work done on culturing crocodile cell lines and cloning the parc gene from Pseudomonas keratitis. Primary crocodile cell lines were established from various organs and immortalized using hTERT. The parc gene was cloned from mutant and wild-type Pseudomonas strains and will be expressed and crystallized to study its role in quinolone antibiotic resistance.
Sequencing the transcriptome reveals complex layers of regulation, Department...Copenhagenomics
This document summarizes a study that analyzed gene expression and regulation in adipose tissue from obese and non-obese individuals. MicroRNA expression was found to be different between the two groups, with many miRNAs downregulated in obesity. One miRNA in particular, miR-193b, was shown to regulate secretion of the inflammatory factor CCL2. Motif activity response analysis identified transcription factors with significantly different activity between obese and non-obese individuals. Together, the results provide new insights into the perturbed transcriptional regulation of adipogenesis and inflammation in human obesity.
This study aimed to clone homologous genes of SND1, a key regulator of secondary cell wall biosynthesis, from Populus trichocarpa. The researcher amplified four SND1 homologs from P. trichocarpa cDNA using PCR. The amplified genes were cloned into E. coli and sequenced. Two colonies were found to contain the correct SND1 sequence insert, while others were false positives. Further work will express and quantify the proteins and determine their effects on other genes and phenotypes.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
The document discusses various molecular cytogenetics techniques including polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and fluorescent in situ hybridization (FISH). It provides details on the principles, techniques, and applications of PCR and RT-PCR. PCR is described as a technique that amplifies specific DNA regions, allowing minute quantities to be analyzed. Key steps involve DNA denaturation, primer annealing, and fragment extension. RT-PCR involves first converting RNA to cDNA then amplifying a specific region. Both techniques have numerous diagnostic and research applications.
olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
This document describes a real-time PCR array for simultaneously evaluating the expression of multiple cytokine mRNAs. The RT2Profiler PCR Array demonstrated high reproducibility, specificity, efficiency, sensitivity, and linear dynamic range. Using this array, 29 genes were found to have at least a 5-fold change in expression between resting and stimulated peripheral blood mononuclear cells after 6 hours of stimulation. The array provides a reliable tool for profiling cytokine pathway gene expression.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The discovery of circulating tumor DNA (ctDNA) in blood, urine
and other bodily fluids has led to a new type of non-invasive
method of characterizing cancer-causing mutations, the liquid
biopsy. With NGS technologies becoming increasingly
sensitive, down to a Limit of Detection (LOD) of 0.1%, they are
rapidly gaining traction as a valid assay for cancer genotyping
and have potential to direct cancer treatment plans. The wideangle
view provided by NGS panels, combined with digital
PCR’s zoomed-in precision detection of DNA provide a
comprehensive picture of a cancer’s genetic makeup. By
applying these complementary techniques at the appropriate
time based on the disease type and stage, cancer treatment
becomes quicker, more precise and more cost-effective in the
future. NGS and digital PCR (dPCR) together provide a
complete picture of the cancer genome.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
A novel method for building custom ampli seq panels using optimized pcr primers Thermo Fisher Scientific
AmpliSeq™ is a next generation sequencing library preparation method for targeted re-sequencing that utilizes highly multiplexed PCR to amplify regions of interest. A key to successful AmpliSeq libraries is the primer panel used for target amplification. Until now primers have been available as pre-assembled ready-to-use panels, or as custom made-to-order panels. We describe a new process for creating customized panels consisting of optimized and verified PCR primers. The primer sets are available as whole genes (i.e., all of the primers needed to create libraries that cover the entire coding regions of genes) and are selectable on the ampliseq.com website by either uploading gene lists or choosing genes from disease research areas.
We show NGS sequencing data from 10 disease research-oriented panels, including newborn screening research and inherited cancer research, assembled from individual pre-verified gene sets. Panel performance data include coverage uniformity, reproducibility, and sensitivity and positive predictive value of variant calling. To demonstrate flexibility of panel content and performance, the coverage uniformity of the 59 genes recommended by the American College of Medical Genetics and Genomics for reporting of incidental findings (ACMG59) was evaluated by themselves and with up to 135 additional genes and shown to be ≥ 97% in all contexts. We also demonstrate the robustness of this method using a variety of sample types (fresh, frozen, and dried blood, cheek swabs) with both manual and fully automated library preparation methods. For Research use only. Not for use in diagnostic procedures.
The document summarizes several biology lab experiments conducted by the author:
1. They performed DNA extraction from samples, PCR, and Western blot techniques. For DNA extraction, their sample did not produce the expected results.
2. They learned aseptic technique and used Gram staining to identify bacteria samples as gram positive or negative.
3. PCR was used to amplify genomic DNA between primers over multiple cycles. Controls were included.
4. Nested PCR with more specific primers was used to further amplify portions of DNA. Exonuclease treated samples before nested PCR.
5. Gel electrophoresis separated DNA fragments by size. PCR products from two plant samples were analyzed, with one showing bands.
Western Blotting Of Camkii Β And T 287Beth Salazar
1. Tomato production is affected by various bacterial, fungal and viral diseases which can cause considerable yield losses.
2. One of the most devastating diseases is tomato leaf curl disease (ToLCD), caused by geminiviruses, which is increasing worldwide and poses a major constraint to tomato production in India.
3. ToLCD causes serious yield losses according to studies from the 1940s and more recently. Effective management strategies are needed to control this and other diseases threatening tomato production.
Hotspot mutation and fusion transcript detection from the same non-small cell...Thermo Fisher Scientific
The presence of certain chromosomal Header
rearrangements and the subsequent fusion
gene derived from translocations has been
implicated in a number of cancers. Hundreds of
translocations have been described in the
literature recently but the need to efficiently
detect and further characterize these
chromosomal translocations is growing
exponentially. The two main methods to identify
and monitor translocations, fluorescent in situ
hybridization (FISH) and comparative genomic
hybridization (CGH) are challenging, labor
intensive, the information obtained is limited,
and sensitivity is rather low. Common sample
types for these analyses are biopsies or small
tumors, which are very limited in material
making the downstream measurement of more
than one analyte rather difficult; obtaining
another biopsy, using a different section or
splitting the sample can raise issues of tumor
heterogeneity. The ability to study mutation
status as well as measuring fusion transcript
expression from the same sample is powerful
because you’re maximizing the information
obtained from a single precious sample and
eliminating any sample to sample variation.
Here we describe the efficient isolation of two
valuable analytes, RNA and DNA, from the
same starting sample without splitting, followed
by versatile and informative downstream
analysis. This methodology has been applied to
FFPE and degraded samples as well as fresh
tissues, cells and blood. DNA and RNA were
recovered from the same non-small cell lung
adenocarcinoma sample and both mutation
analysis, as well as fusion transcript detection
was performed using the Ion Torrent PGM™
platform on the same Ion 318™ chip. Using
10ng of DNA and 10ng of RNA input, we
applied the Ion AmpliSeq™ Colon and Lung
Cancer panel to analyze over 500 COSMIC
mutations in 22 genes and the Ion AmpliSeq™
RNA Lung Fusion panel to detect 40 different
fusion transcripts.
This document provides an overview of polymerase chain reaction (PCR). It discusses:
1. The key discoveries and scientists that developed PCR, including Kary Mullis who won the 1993 Nobel Prize for his conception of PCR.
2. The basic steps and components of PCR including DNA template, primers, Taq polymerase enzyme, repeated temperature cycling of denaturation, annealing and extension to exponentially amplify the target DNA region.
3. Applications of PCR including DNA cloning, medical diagnostics, forensics, and research. PCR is used to amplify specific genes or DNA regions for various downstream analyses and applications.
Universal and rapid salt extraction of high quality genomic dna for pcr-based...CAS0609
This document describes a simple and universal method for extracting high-quality genomic DNA from a variety of organisms including plants, fungi, insects, and shrimp. The method uses a salt-based homogenizing buffer and SDS to extract DNA from as little as 50mg of fresh tissue. The extracted DNA is of sufficient quality and quantity to be used in PCR, restriction digestion, and other molecular techniques. The method is fast, inexpensive, and does not require expensive equipment, making it suitable for laboratories with limited resources. Test results demonstrated the method successfully extracted high molecular weight DNA from many diverse organisms without modification, indicating its universal applicability.
Through genetic barcoding of the tufA gene, an unknown green algae specimen was identified as belonging to the Ulva compressa clade. DNA was isolated from the specimen and the tufA gene was amplified via PCR. The gene was cloned and the nucleotide sequence analyzed and compared to sequences in GenBank. Phylogenetic analysis indicated the specimen was most similar to Ulva sp. BER-2007, but could not be identified to the species level. While in the U. compressa clade, further testing is needed to confirm its identity as a potential new species of Ulva in Narragansett Bay.
Next generation sequencing of the whole transcriptome enables high resolution measurement of gene expression activity in different tissue and cell types. This methodology provides an in depth study of known transcripts and depending on the data analysis, allows identification of additional transcript types such as transcript variants, fusion transcripts, and small and long ncRNAs.
In this study we performed RNA-Seq using the Ion Torrent™ sequencing platform to compare the expression profile of testicular germ cell cancers (seminoma type, n=3) and normal testis (n=3). Using Partek Flow® 3.0 and TopHat/BowTie or Star aligners, we aligned the reads to the human genome and mapped sequences to the RefSeq database. Differentially expressed genes were identified and screened with additional germ cell tumors.
PCA analysis showed clear separation of the two sample types indicating biological differences. List of differentially expressed genes generated from TopHat/Bowtie and Star were similar. We identified a large number of genes that were up and down regulated with high degree of significance (p<0.01,>2X FC (fold change)). These included genes related to testicular tissue type, stem cell pluripotency (NANOG; POU5F1) and proliferation (KRAS, CCND2).
In addition, a number of differentially expressed noncoding RNAs were identified (SNORD12B, XIST). The method was validated on a small set of genes (n=20) using qPCR (TaqMan® Assays) and were found to be correlated. We used the OpenArray® platform to quickly and quantitatively screen 102 differentially expressed genes and 10 endogenous control genes across a number of different testicular germ cell cancer types.
We used a complete work flow solution from sample prep to NGS to qPCR to compare the expression profile of normal testis and seminoma type germ cell tumors. From the NGS experiments we identified a large number of differentially expressed genes for qPCR screening with samples from different types of germ cell tumors. Results from these screening studies will be presented.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Le...Agriculture Journal IJOEAR
Abstract—ACLSV is one of the major fruit viruses and can cause severe diseases in species of family Rosaceae. Previous RT-PCR methods are available to detect ACLSV in hawthorn samples, but not to evaluate the infected level of ACLSV. In this study, a TaqMan-based quantitative RT-PCR detection method targeting CP gene of ACLSV was first established and the sensitivity and reproducibility were investigated. The results indicated that this standard curve between log of plasmid DNA concentration versus the cycle threshold (Ct) value generated a linear fit with a linear correlation (R2) of 0.99 and the PCR efficiency was more than 90%. The quantitative RT-PCR method was high sensitive and able to detect 6.9 × 102 copies•μL-1 of ACLSV RNA. Compared with the conventional RT-PCR method, it was 100-fold sensitive in detection of ACLSV. In addition, different organs of hawthorn samples were examined using the quantitative RT-PCR repeatedly and the result revealed that the quantitative RT-PCR is not only an effective detection method, and can obtain an absolute quantitation for ACLSV.
Comparing Mutation Detection Sensitivity from Matched FFPE Tissue and Liquid ...Thermo Fisher Scientific
Cancer researchers are avidly working to enable circulating cell free DNA (cfDNA) profiling as a new more sensitive tool to detect and screen for the presence of solid tumors before detection through clinical methods. Despite the high level of interest in cfDNA, researchers still have reservations until enough data has demonstrated complementarity between methodologies. In this study, we examined the data quality and concordance of mutations called for a small number of matched formalin fixed paraffin embedded (FFPE) tissue and plasma samples.
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.
The document summarizes research analyzing two proteins, AvrGf1 and AvrGf2, that are crucial for the bacterial pathogen Xanthomonas citri to infect citrus plants. Genetic analysis identified the genes for these proteins. Sequence analysis found motifs indicating the proteins are injected into chloroplasts and interact with a citrus cyclophilin after a conformational change. The goal is to express and purify the proteins to study their interaction with the cyclophilin and understand how it activates them during infection.
The experiment extracted, cloned, and sequenced the GAPC gene from thyme plant DNA inserted into E. coli plasmid vectors. Nested PCR and gel electrophoresis showed the thyme DNA was suitable for cloning. Restriction digest confirmed the E. coli plasmids accepted the GAPC insert. Sanger sequencing determined the plasmid sequences, which BLAST analysis found were nearly identical to the known GAPC gene sequence, indicating the experiment successfully cloned and sequenced the thyme GAPC gene.
This document summarizes a study that used DNA analysis of botanical evidence found at crime scenes to help solve criminal cases. DNA was extracted from plant evidence using a CTAB method. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was performed using random primers to develop DNA profiles of the standard and suspected samples. In two cases, the RAPD-PCR profiles matched between the standard and suspected samples, suggesting they came from the same location. In one case, the profiles did not match. The study demonstrates how DNA analysis of botanical evidence can help determine if a victim and crime scene are linked or differentiate between potential crime scenes.
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase
Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA
Simultaneous Isolation of RNA & DNA from one FFPE SampleQIAGEN
The document summarizes the AllPrep DNA/RNA FFPE Kit, which simultaneously isolates genomic DNA and total RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. FFPE samples are lysed and centrifuged to separate the RNA-containing supernatant from the DNA-containing pellet. The supernatant and pellet are then processed separately to purify high-quality RNA and DNA suitable for downstream applications like real-time PCR and pyrosequencing. Results show the kit yields RNA and DNA of sufficient quality and quantity from old FFPE samples for gene expression analysis and detection of genetic mutations.
This document summarizes a presentation given by Minoru Kubo on single cell analyses for plant reprogramming studies. The presentation covered how next generation sequencing and single cell analyses have changed biological research. It discussed single cell transcriptomics for analyzing plant reprogramming at the single cell level. The goal is to identify reprogramming genes, understand cell-cell interactions during reprogramming, and validate theories of reprogramming using the moss Physcomitrella patens as a model organism. Positioning information from single cell analyses could provide new insights into cell lineages, interactions, and multicellular development.
Similar to suraj_jaladanki_examining_Malaclemys_terrapin_genome_scaffolds (20)
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
1. Suraj Jaladanki
FIRE 155
Sequencing of the Diamondback Terrapin’s Genome and its Future Applications
Introduction
There are a multitude of attributes which are exclusive to turtles, making them a unique
group to study. For one, the dorsal portion of chelonian shells, or the carapace, is formed through
vertebrae and ribs (Wang, 2013). Turtles were phylogenetically shown to be morphologically
conservative, with their distinct body formation dating back at least 210 million yeas (Shaffer,
2013). In addition, testudines have extremely long lifespans, many of which are reproductively
active at later stages in life (Shaffer, 2013). Other significant features of turtles are their abilities
to survive in both anoxic and freezing conditions, while encountering minimal tissue damage
(Shaffer, 2013).Wang et al. found that turtle-specific pattern formation occurred to create the
shell (Wang, 2013). Shaffer et al. studied the genome of the western painted turtle (Chrsemys
picta bellii), and found potential genes involved in the species’ capacities to resist extreme
anoxia and freezing temperatures (Shaffer, 2013).
The diamondback terrapin (Malaclemys terrapin) is another unique chelonian species
which has not yet been thoroughly studied. It is believed to be one of the few turtles in the world
that lives solely in brackish water, estuaries, and lagoons, and examining these attributes in
terrapins could explain the genetic basis for terrapin’s capacity to live in this environment
(Basic). However, a major impediment limiting the progress of terrapin genomic studies is that
the terrapin genome has not been completely sequenced. In testudine studies, genomic analysis is
a vital component in research, as it provides scientists a means of comparison for examining the
similarities and differences of two species at a genetic level, known as comparative genomics
(Genome). Although genes account for less than 25% of the DNA in the genome, understanding
2. the full sequence aids in determining the roles of non-coding nucleotide regions (Genome). After
the human genome was completely sequenced after thirteen years of work, scientists discovered
that human have approximately 20,000-25,000 genes as opposed to the predicted 100,000 genes,
an interesting result suggesting that a majority of the genome does not directly contribute to
genes (Silverman).
This study details this group’s efforts to sequence the terrapin genome in order to be used
as a launching point for future terrapin analyses. The terrapin genome has been sequenced;
however, the programs used in sequencing are unable to produce a completed genome due to
gaps. Since the latest technology has not been fully successfully in filling all gaps in the terrapin
genome, the team reverts to older methods of PCR to close gaps in the terrapin genome.
Sequencing the diamondback terrapin’s genome in particular will assist in determining how large
the terrapin genome is (in base pairs) and examining the evolutionary history between terrapins
and other chelonian species. Knowledge will be added to the work of testudine studies, leading
to a more comprehensive understanding of turtle species, aiding scientists in using turtle
properties and mechanisms to heal human ailments including cardiac infarctions and cerebral
strokes.
Materials and methods:
Sequencing and Primer Creation
One adult female M. terrapin (Diamondback terrapin) was found on Poplar Island, 2
miles off the coast of Sherwood, Maryland, and was collected along with her 15 eggs. DNA and
RNA were isolated from all organismal tissues, including blood. Through a sequencing program,
the majority of the terrapin’s DNA scaffolds were reassembled. The unknown nucleotide regions
3. were then analyzed to determine if the gaps were between 100-800bp as this is the optimal size
of a DNA strand that can be visualized on a gel.
A portion of the genetic data, specifically the terrapin DNA isolated from blood, was
analyzed in this study. First, the scaffolds were compared with GenBank sequences to note if
similarities existed between the terrapin genome sequence and GenBank non-redundant
sequences, and this was accomplished through the Nucleotide Blast program which is a part of
the NCBI BLAST. After these similarities were noted, the scaffolds were utilized to design
specific PCR Primers. Utilizing the program NCBI Primer-Blast (Primer 3), primers were
designed to fill the gap, with the primer ranges based on the length of the un-sequenced gaps to
ensure that the entire gap would be copied. The primers, forward and reverse, were then ordered
for each scaffold from Integrated DNA Technologies (IDT).
Once the PCR primers were attained, a 100uM stock was created. This was accomplished
by spinning the tubes, and adding the amount of TE buffer (pH 7.4, Fluka) needed to make
100uM stock. Several tubes of working 10x stock were created through diluting 1:10 in TE
buffer. The 100 uM stock and 10x stocks were placed in a -20o
C freezer until further needed.
PCR Protocol
PCR was implemented to amplify the amount of scaffolds to be sequenced at a later time.
Eight labeled DNAse free PCR Eppendorf tubes (0.2mL) were used to amplify three separate
scaffolds in the first three tubes. The fourth tube acted as a positive control, and tubes 5-8 served
as negative controls for the three sets of PCR Primers (10x) in tubes 5-7 and the positive control
in tube 8. The reaction volume for each tube was 50uL. PCR mixes contained a concentration of
1x GoTaq Green (Promega, Cat No. M7122, Lot# 0000140255), forward and reverse primers
4. (1.ouM) and genomic terrapin DNA (1-2 ng/uL, isolated from blood), and nuclease-free water
(Ambion).
These tubes were added to the thermo-cycler (BioRad iCycler, V.3.021) which was
already pre-heated to 95o
C. PCR was performed under standard conditions (95o
C for 2 minutes,
35 cycles at 95o
C for 15 seconds, 55o
C for 30 seconds, and 72o
C for 60 seconds followed by a
final elongation at 72o
C). Upon completion, the temperature remained at a constant 4o
C. PCR
was optimized by increasing the annealing temperature to 57o
C instead of 55o
C, with all other
conditions remaining the same. After the tubes were retrieved, they were stored at 4o
C until
further needed.
Gel Electrophoresis Protocol
The electrophoresis procedure was utilized after PCR to visualize the size of the scaffold
through comparison with a 1KB DNA Ladder. The PCR products were subjected to agarose gel
electrophoresis (1% in 1x TAE, BioRad Gel Electrophoresis Unit Sub Cell GT, 90 V/cm.) and
staining with ethidium bromide. A 1 kb DNA Ladder was created through mixing 6uL of a 1KB
DNA ladder (New England Biolabs, Cat#N232L, Lot#1251411), 14uLTE buffer, and 5uL gel
loading dye (6x, NEB) to estimate PCR product sizes.
The gel was run for 45 minutes at 90V and at 400mA. DNA bands were visualized
(EpiChem 3 Darkroom) and photographed. These results were then compared to the size
predicted by the sequencing program. After initial outcomes, the protocol was modified to create
a 2% gel which contained 0.90g of agarose for the purpose of forming higher resolution gels, and
the remaining procedures were followed in the manner stated previously.
DNA Purification Protocol
5. Once the sizes of the scaffolds were confirmed, the DNA attained in PCR was needed to
be purified to be sent for sequencing. This DNA was purified using Qiagen Quickspin columns
according to the manufacturer’s recommendations. Buffer PE served as the wash buffer, and
Buffer PB acted as the binding buffer. This solution in a QIAquick column was then washed
with Buffer PE added to the QIAquick column. DNA was eluted with elution buffer (10 mM
Tris-Cl, pH 8.5) to the QIAquick membrane. Results of DNA purification were visualized by
running a gel (2% in 1x TAE, 90 V/cm) with 10uL of purified DNA and 2uL of loading dye, to
estimate DNA concentration, with the remaining purified DNA stored at 4o
C. Once the sample
was calculated to have a DNA concentration between 5-40 ng/uL, it would be used in
sequencing. If the concentration was above the limit, it could be diluted to lower the DNA
concentration to an adequate level.
Sequencing of Scaffolds
Once the scaffolds were confirmed to have the same size both before and after
purification through analysis of gel electrophoresis results, the purified DNA was sent to the
sequencing company, Macrogen.
6. Results
Figure I
(i) PCR Products of 1% Gel Electrophoresis Results at 55o
C annealing temperature
(ii) PCR Products are arranged in the gel as follows: (Lane 1) scaffold00227, (Lane 2)
scaffold00228, (Lane 3) scaffold00041, (Lane 4) positive control H gene: rp, (Lane 5)
negative control scaffold00227 w/out Terrapin DNA, (Lane 6) negative control
scaffold00228 w/out Terrapin DNA, (Lane 7) negative control scaffold00041 w/out
Terrapin DNA, (Lane 8) negative control of H gene w/out Terrapin DNA, (Lane 9)
1KB Ladder
Figure II
(i) PCR Products of 1% Gel Electrophoresis Results at 57o
C annealing temperature
7. (ii) PCR Products are arranged in the gel as follows: (Lane 9) 1KB Ladder, (Lane 10)
scaffold00227, (Lane 12) scaffold00041, (Lane 13) positive control H gene: rp, (Lane
14) negative control scaffold00227 w/out Terrapin DNA, (Lane 15) negative control
scaffold00041 w/out Terrapin DNA, (Lane 16) negative control of H gene w/out
Terrapin DNA
Figure III
(i) 2% Gel Electrophoresis of the purified PCR products of the above PCR products
scaffold00227 and scaffold 00041
(ii) The Gel is arranged as follows: (Lane 9) 1KB Ladder, (Lane 10) scaffold00227,
(Lane 11) scaffold00041,
Figure IV
8. (i) PCR Products of 2% Gel Electrophoresis Results at 55o
C annealing temperature
(ii) PCR Products are arranged in the gel as follows: (Lane 9) 1KB Ladder, (Lane 10)
scaffold00219, (Lane 11) scaffold00225, (Lane 12) scaffold00229, (Lane 13)
scaffold00228, (Lane 14) positive control D gene: rp, (Lane 15) negative control
scaffold00219 w/out Terrapin DNA, (Lane 16) negative control scaffold00225 w/out
Terrapin DNA, (Lane 17) negative control scaffold00229 w/out Terrapin DNA, (Lane
18) negative control scaffold00228 w/out Terrapin DNA, (Lane 19) negative control
of D gene w/out Terrapin DNA
Scaffold ID Expected Length
(Computer-
determined Gap
Length)
Observed Length
on First Gel
Observed Length
on Second Gel
Purified PCR
Product Lengths
Scaffold 00227 821bp Not seen <500bp <500bp
Scaffold 00228 841bp <500bp Not run Not run
Scaffold 00041 888bp <500bp <500bp <500bp
Positive Control
H (gene rp)
631bp <500bp 750bp Not run
Figure V
(i) Summary of Results from Sequencing First Three Scaffolds (scaffold 00227, scaffold
00228, scaffold00041). Scaffold 00228 was not attained from PCR due to loss of
product.
Scaffold ID Expected Length
(Computer-
determined Gap
Length)
Observed Length
on First Gel
Scaffold 00219 956bp <500bp
Scaffold 00225 930bp ~500bp
Scaffold 00229 937bp <500bp
Scaffold 00228 841bp <500bp
Positive Control D (gene rp) 723bp <750bp
Figure VI
(i) Summary of Results from Sequencing Second Set of Scaffolds and Previous Scaffold
(scaffold 00219, scaffold 00225, scaffold00229, scaffold 00228)
9. Figure VII
(i) Sequencing Results of Scaffold 00041 from Macrogen
Figure VIII
(i) Sequencing Results of Scaffold 00219 from Macrogen
10. Figure IX
(i) Sequencing Results of Scaffold 00225 from Macrogen
Discussion
For the first three scaffolds (scaffolds 00227, 00228, 00041), the results shown in Figure I
appeared significantly less than expected. This was known because the positive control H which
was expected to be 631bp appeared less than 500bp, and this was not seen in the results of other
researchers, indicating an issue only in this gel run. The lack of proper size placement of the
positive control invalidated the results of this run, and these lower than expected results most
likely appeared because of an operation through loading the incorrect samples in wells. A band
did not appear in Well 1 because the sample could have leaked out when it was loaded and other
forms of user error.
In the next gel analysis, PCR was performed at a 57o
C annealing temperature instead of the
original 55o
C temperature because it was possible that un-optimized annealing temperatures led
11. to the incorrect binding of primers, leading to smaller than expected sized bands appearing in the
gel electrophoresis. These results are seen in Figure II where all loaded samples appeared. The
PCR tube containing scaffold00228 popped during PCR leading to a major loss of sample and
inability to load it in the gel. These results could be used in the analysis because the positive
control H appeared closer to its predicted value of 631bp, appearing at 750bp.The change in
annealing temperature could have had an impact on the sizes of the bands seen in the gel, so this
annealing temperature was continued to be used in future runs of PCR. Scaffolds 00041 and
00027 still were shown to be less than 500bp compared to their respective predicted sizes of
888bp and 821bp respectively. The PI noted that the computer programs utilized to predict these
sizes could be inaccurate to a degree of several hundred base pairs, so the expected sizes should
not have much weight in future analysis of results seen in gels.
After DNA purification, the results were not seen in a 1% gel, and this could have occurred
as a result of the lower resolution of the 1% gel. As a result, a 2% gel was created and used to
run the purified samples of scaffolds 00227 and 00041. With this agarose concentration, faint
bands were visible for both scaffolds as seen in Figure III, and they again appeared lower than
500bp, closely matching their respective sizes in their pre-purified forms. Since both pre- and
post- purification results were similar, the concentrations of the DNA in the scaffolds were
determined by comparison to the DNA ladder. The concentrations for scaffolds 00227 and 00041
were 7.5ng/uL and 15ng/uL respectively. This was well within the range of 5-40ng/uL as
requested by the DNA sequencing company, so the tubes containing these scaffolds were sent for
sequencing. Since there was no internal primer for scaffold 00227, it could not be sequenced, so
only scaffold 00041 was able to be sequenced.
12. With the next set of primers from IDT that were created through Primer-BLAST, the DNA
was amplified through PCR and run on the gel, as seen in Figure IV. The new scaffolds 00219,
00225, 00229 were used in PCR. In addition, scaffold 00228, which was unable to be purified
with the first set of samples, was included with this set. All bands appeared less than 500bp, but
the positive control D which had an established size of 723bp appeared at 750bp, validating the
results of the run. When comparing the predicted sizes to the observed sizes, there were
differences of at least 300bp, and the expected results were pointed out earlier to be inaccurate.
All lanes had single bands except for lane containing scaffold 00229 which had a double band.
This double band is possible evidence of a primer dimer, which meant that this sample couldn’t
be purified because there were multiple segments containing this sequence. Thus, all samples
other than scaffold00229 were purified to be compared with the results seen in Figure IV.
The purified samples of scaffolds 00219, 00225, and 00228 were visualized in Figure V.
The bands seen were close to their respective bands seen in the unpurified DNA samples in
Figure IV, with all bands appearing less than the 500bp band, but in close proximity to it. Since
the results in Figure V more or less matched the data in Figure IV, the concentrations of DNA in
the samples were calculated and were found to all be 50ng/uL, for scaffolds 00219, 00225, and
00228. Although the concentrations were over the sequencing company’s range of 5-40ng/uL, it
was found that using the brightness of the bands to calculate DNA concentration could lead to
slightly inaccurate results, and a concentration marginally over the recommended limit was
sufficient to be used in sequencing.
The results of the sequenced scaffold 00041 returned and are seen in Figure VII. The
chromatogram showed numerous uneven spacing between the bands and many mixtures of
colors at one band location, indicating unusable results. The QV scores also confirmed that the
13. data for scaffold00041 could not be used in the terrapin genome’s sequencing process because
there were only 5 QV scores which were >= 16 and 4 QV scores which were >= 20, which
demonstrates low confidence in the created sequence. The poor results seen with scaffold 00041
could have arisen due to a significant amount of noise being present, mis-called nucleotides, and
double peaks arising from single nucleotide polymorphisms (SNP’s). Collectively, these factors
lead to this scaffold unable to be sequenced with this particular set of primers, and this scaffold
will have to be approached in the future with a different set of primers in the hopes of attaining
more promising results in the sequencing process. The three other scaffolds (00219, 00225, and
00228) contain internal primers, so they were sent to the sequencing company.
The sequence for scaffold 00225 was returned from Macrogen and is seen in Figure VIII.
Similar to the results seen in Figure VII, there were numerous mis-spaced nucleotides and a
moderate level of noise throughout the chromatogram. Although the QV scores were not able to
be found, they most likely contain low numbers for bases which were >=16 and >=20 due to the
level of noise and mis-spaced nucleotides present. These features of the chromatogram lead this
scaffold to be unable for sequencing using this set of primers.
Also, the sequence for scaffold 00229 was returned from Macrogen and is seen in Figure
IX. Analogous to the results seen in other chromatograms (Figures VII and VIII), numerous mis-
spaced nucleotides and a moderate level of noise were present throughout the chromatogram.
The QV scores were not able to be located, but they most likely provide low values above the 16
and 20 thresholds, given the noise and mis-spaced nucleotides. These features of the
chromatogram lead this scaffold to be unable for sequencing using this set of primers.
14. References:
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http://www.defenders.org/diamondback-terrapin/basic-facts
(2003, January 15). GENOME SEQUENCING. Genome News Network. Retrieved from
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Rogers, Y.C., Munk, A.C., Meincke, L.J., Han, C.S. (2005). Closing bacterial genomic sequence
gaps with adaptor-PCR. BioTechniques, Volume 39 (1), pp. 31-34.
Shaffer B., et al. (2013). The western painted turtle genome, a model for the evolution of
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Silverman, J. What have we learned from the Human Genome Project? Retrieved from
http://science.howstuffworks.com/life/genetic/human-genome-project-results1.htm
Wang, Z., Pascual-Anaya, J., Zadissa, A., Li, W., Niimura, Y., Huang, Z., Li, C., White, S.,
Xiong, Z., Fang, D., Wang, B., Ming, Y., Chen, Y., Zheng, Y., Kuraku, S., Pignatelli, M.,
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