- Currently, HLA typing for stem cell transplants can be determined from DNA samples, but Rh blood group antigens still require serological testing of blood samples.
- This project aims to develop a targeted next-generation sequencing assay to determine RhD and RhCE antigens directly from DNA samples, which could streamline the donor matching process.
- The researchers optimized long-range PCR and gene-specific indexing to amplify and sequence RhD and RhCE from DNA despite their high sequence similarity. Preliminary results identified the patient's Rh antigens as D-positive, homozygous C, and homozygous e directly from DNA sequencing.
- If validated, this approach could allow Rh antigen typing from the same DNA samples
Genetic engineering lecture PPT- Dr. Mangesh Dagwal 2020Dr.Mangesh Dagawal
I have prepared this lecture presentation on the topic Genetic Engineering for our students and also delivered guest lecture on this topic at various colleges in our region.
SBVRLDNACOMP:AN EFFECTIVE DNA SEQUENCE COMPRESSION ALGORITHMijcsa
There are plenty specific types of data which are needed to compress for easy storage and to reduce overall retrieval times. Moreover, compressed sequence can be used to understand similarities between biological sequences. DNA data compression challenge has become a major task for many researchers for the last few years as a result of exponential increase of produced sequences in gene databases. In this research paper we have attempt to develop an algorithm by self-reference bases; namely Single Base Variable Repeat Length DNA Compression (SBVRLDNAComp). There are a number of reference based compression methods but they are not satisfactory for forthcoming new species. SBVRLDNAComp is an optimal solution of the result obtained from small to long, uniform identical and non-identical string of nucleotides checked in four different ways. Both exact repetitive and non-repetitive bases are compressed by SBVRLDNAComp.The sound part of it is without any reference database BVRLDNAComp achieves 1.70 to 1.73 compression ratio α after testing on ten benchmark DNA sequences. The compressed file can be further compressed with standard tools (such as WinZip or WinRar) but even without this SBVRLDNAComp outperforms many standard DNA compression algorithms.
Cloning vector
is a small piece of DNA Allow the foreign DNA inserted inside cell
Plasmid
Bacteriophage
Cosmid
BAC
YAC
HAC
Plasmid
Bacteriophage
Cosmid
BAC
YAC
HAC
CRISPR/Cas9 gene editing is based on a microbial restriction system, that has been harnessed for genome targeting using only a short sequence of RNA as a guide.
Characterization of RNA binding protein RBPMS as a novel marker for retinal ...Lior Schenk
Lior I. Schenk1, Praseeda Venugopalan1,2, Xiong Zhang1, and Jeffrey L. Goldberg1,3
1 University of California, San Diego, La Jolla, CA; 2 University of Miami, Miami, FL; 3 UCSD Shiley Eye Center, La Jolla, CA
Bioo Scientific - Absolute Quantitation for RNA-SeqBioo Scientific
Accurate quantitation is a critical issue for most RNA-Seq analysis. As the efficiency of PCR amplification is sequence-dependent, with some transcripts being preferentially amplified over others, PCR amplification introduces bias during NGS library construction. The traditional approach to removing these artifacts, introduced during PCR, involves removing all fragments with identical start and stop sites. However, detailed analyses have shown that many original fragments have identical start and stop sites, and this method can incorrectly eliminate unique fragments from NGS data, thus reducing its accuracy. Bioo Scientific has incorporated Molecular Indexes™ into its NEXTflex™ Rapid Directional qRNA-Seq™ Kit, allowing for a more accurate resolution of duplicates introduced during PCR amplification. This presentation describes how Molecular Indexes can be used to increase the accuracy of RNA-Seq analysis.
Bioo Scientific - Simplify and Reduce Cost of mtDNA Isolation and Library PrepBioo Scientific
Mutations in mitochondrial DNA (mtDNA) have been implicated in various human disorders and in aging, making NGS analysis of mtDNA a priority for a number of labs. However, accurately determining the diversity of mtDNA has been difficult for a number of reasons. The standard methods for mitochondrial DNA extraction have a number of limitations making them inferior solutions for NGS library preparation. Bioo Scientific has commercialized a kit which overcomes these limitations of mtDNA isolation by selectively digesting linear nuclear DNA (nDNA) while leaving circular mtDNA intact. This technology has been incorporated into the NEXTflex mtDNA-Seq Kit which includes optimized reagents for the isolation of mtDNA and for the construction of Illumina mtDNA libraries. Libraries constructed using the NEXTflex mtDNA-Seq Kit are ideal for many NGS applications including heteroplasmy analysis.
Cold Spring Harbor. Single Cell Analyses Meeting. November 11 - 14, 2015. Slides for talk: PAGODA—Pathway and gene set overdispersion analysis characterizes single cell transcriptional heterogeneity.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
Genetic engineering lecture PPT- Dr. Mangesh Dagwal 2020Dr.Mangesh Dagawal
I have prepared this lecture presentation on the topic Genetic Engineering for our students and also delivered guest lecture on this topic at various colleges in our region.
SBVRLDNACOMP:AN EFFECTIVE DNA SEQUENCE COMPRESSION ALGORITHMijcsa
There are plenty specific types of data which are needed to compress for easy storage and to reduce overall retrieval times. Moreover, compressed sequence can be used to understand similarities between biological sequences. DNA data compression challenge has become a major task for many researchers for the last few years as a result of exponential increase of produced sequences in gene databases. In this research paper we have attempt to develop an algorithm by self-reference bases; namely Single Base Variable Repeat Length DNA Compression (SBVRLDNAComp). There are a number of reference based compression methods but they are not satisfactory for forthcoming new species. SBVRLDNAComp is an optimal solution of the result obtained from small to long, uniform identical and non-identical string of nucleotides checked in four different ways. Both exact repetitive and non-repetitive bases are compressed by SBVRLDNAComp.The sound part of it is without any reference database BVRLDNAComp achieves 1.70 to 1.73 compression ratio α after testing on ten benchmark DNA sequences. The compressed file can be further compressed with standard tools (such as WinZip or WinRar) but even without this SBVRLDNAComp outperforms many standard DNA compression algorithms.
Cloning vector
is a small piece of DNA Allow the foreign DNA inserted inside cell
Plasmid
Bacteriophage
Cosmid
BAC
YAC
HAC
Plasmid
Bacteriophage
Cosmid
BAC
YAC
HAC
CRISPR/Cas9 gene editing is based on a microbial restriction system, that has been harnessed for genome targeting using only a short sequence of RNA as a guide.
Characterization of RNA binding protein RBPMS as a novel marker for retinal ...Lior Schenk
Lior I. Schenk1, Praseeda Venugopalan1,2, Xiong Zhang1, and Jeffrey L. Goldberg1,3
1 University of California, San Diego, La Jolla, CA; 2 University of Miami, Miami, FL; 3 UCSD Shiley Eye Center, La Jolla, CA
Bioo Scientific - Absolute Quantitation for RNA-SeqBioo Scientific
Accurate quantitation is a critical issue for most RNA-Seq analysis. As the efficiency of PCR amplification is sequence-dependent, with some transcripts being preferentially amplified over others, PCR amplification introduces bias during NGS library construction. The traditional approach to removing these artifacts, introduced during PCR, involves removing all fragments with identical start and stop sites. However, detailed analyses have shown that many original fragments have identical start and stop sites, and this method can incorrectly eliminate unique fragments from NGS data, thus reducing its accuracy. Bioo Scientific has incorporated Molecular Indexes™ into its NEXTflex™ Rapid Directional qRNA-Seq™ Kit, allowing for a more accurate resolution of duplicates introduced during PCR amplification. This presentation describes how Molecular Indexes can be used to increase the accuracy of RNA-Seq analysis.
Bioo Scientific - Simplify and Reduce Cost of mtDNA Isolation and Library PrepBioo Scientific
Mutations in mitochondrial DNA (mtDNA) have been implicated in various human disorders and in aging, making NGS analysis of mtDNA a priority for a number of labs. However, accurately determining the diversity of mtDNA has been difficult for a number of reasons. The standard methods for mitochondrial DNA extraction have a number of limitations making them inferior solutions for NGS library preparation. Bioo Scientific has commercialized a kit which overcomes these limitations of mtDNA isolation by selectively digesting linear nuclear DNA (nDNA) while leaving circular mtDNA intact. This technology has been incorporated into the NEXTflex mtDNA-Seq Kit which includes optimized reagents for the isolation of mtDNA and for the construction of Illumina mtDNA libraries. Libraries constructed using the NEXTflex mtDNA-Seq Kit are ideal for many NGS applications including heteroplasmy analysis.
Cold Spring Harbor. Single Cell Analyses Meeting. November 11 - 14, 2015. Slides for talk: PAGODA—Pathway and gene set overdispersion analysis characterizes single cell transcriptional heterogeneity.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
Tema 2: la integración curricular de las TIC Belengisbe
Presentación sobre los cambios en la manera de ver las tecnologías, los nuevos roles que han adquirido tanto los profesores como los alumnos a consecuencia de la utilización de las nuevas tecnologías en el ámbito educativo, y la integración curricular de las TIC en el aula con los niveles que se deben cumplir para que dicha integración sea posible.
Concurrent Determination of ABO RhD Blood Types and the HIV-1 Resistance Mark...Thermo Fisher Scientific
For optimal outcomes when matching bone marrow donors with their recipients, it is preferable to use bone marrow of identical or compatible blood types. Bone marrow registries thus require high resolution HLA genotyping data to match donor specimens with their recipients. We developed a research assay to aid in these investigations, which utilizes buccal swab DNA from potential donors to determine the ABO and Rh-antigen genotypes. In addition, the assay detects a 32 bp deletion in the CCR5 gene. Homozygous carriers of this deletion are resistant to HIV-1 infection, and thus could be valuable stem cell donors for HIV-infected recipients
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
DNA Fingerprinting of plants . History,procedure of DNA fingerprinting, PCR and NON PCR technique like RAPD,SSR,RELPs, application of DNA fingerprinting, advantage and disadvantage of DNA fingerprinting.
DNA sequencing is a laboratory technique used to determine the exact sequence of bases (A, C, G, and T) in a DNA molecule. The DNA base sequence carries the information a cell needs to assemble protein and RNA molecules. DNA sequence information is important to scientists investigating the functions of genes.
In medicine, DNA sequencing is used for a range of purposes, including diagnosis and treatment of diseases. In general, sequencing allows health care practitioners to determine if a gene or the region that regulates a gene contains changes, called variants or mutations, that are linked to a disorder.
DNA sequencing refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate. Establishing the sequence of DNA is key to understanding the function of genes and other parts of the genome. There are now several different methods available for DNA sequencing, each with its own characteristics, and the development of additional methods represents an active area of genomics research.
Validation of rare Variants in the Schizophrenia-linked gene DPYSL2 - Fatuma ...
Abstract (1)
1. Creating a Targeted Next Generation Sequencing Assay to Identify RH antigens from DNA for
Stem Cell Transplants
Abigail Joseph
Principal Investigator: William Lane MD, PhD
Mentors: Peter Tonellato PhD; Helen Mah, MS; John Baronas
Dana-Farber Cancer Institute
Using DNA assays to determine human leukocyte antigen (HLA) type is standard practice when
seeking to find stem cell transplant donor matches. Recently, next generation sequencing
(NGS) has been successfully developed to perform accurate HLA typing. As a result, stem cell
donors are typed using DNA samples collected with cheek swab kits. ABO and Rh mismatches
between donor and recipient can result in major and minor incompatibilities that can delay
erythroid engraftment and require multiple rounds of red blood cell (RBC) transfusions. An ABO
and Rh NGS assay could improve the stem cell transplant matching process because only DNA
samples are collected from prospective donors. However, one major obstacle in using NGS to
determine the presence of Rh antigens from DNA is the similarity between the RHD and RHCE
genes. The objective of our experiment was to determine whether a targeted NGS assay could
be developed to determine the specific Rh antigens in a patient’s RBCs. We performed long
range PCRs to test optimal primer combinations for successful amplification of the genes. Gene
specific indexes were added to the RHD and RHCE PCR products, prior to sequencing, so
identical regions of the genes could be clearly labeled. The results of the sequence alignments
showed that the primers that targeted RHD exons 1-6 and RHCE exons 1-4 resulted in the
largest PCR products while maintaining strong alignment coverage. In addition, the DNA reads
aligned according to our expectations allowing us to predict the patient’s antigen type as D
antigen positive, homozygous for the C antigen, and homozygous for the e antigen. These
findings suggest that Rh antigens can be determined from DNA assays without serologic
testing. Consequently, the same DNA sample used for HLA typing can be used for Rh antigen
typing, thus streamlining the stem cell donor match process.
2. Currently HLA (human Leukocyte Antigen) typing is done completely from DNA
● However, RBC antigens still cannot be determined from DNA and must be confirmed
serologically
● This hinders the process of finding donors for Stem Cell Transplant,
○ Although a DNA sample, such as spit or cheek cells, that are sent in can be used
to determine a potential donor's HLA type, that same sample can’t be used to
determine RBC type
○ Donor must physically come in to donate blood and test must be done
serologically, which is time consuming and expensive
● This is especially relevant for patients with leukemia or Lymphoma who need Stem Cell
Transplants since their immune systems were eliminated in the treatment process
● There is a need to find exact matches or else the patient is at risk for HTR (hemolytic
Transfusion Reaction) or GvHD (Graft vs. Host Disease)
3. ● Purpose is to create a targeted assay that uses Next Generation Sequencing to confirm
RH, RhD and RhCE, antigens on RBC similar to how HLAs are confirmed
● Then theoretically one DNA sample could be used to find the most promising exact HLA
and RBC antigen matches, so only a few would have to be brought in for serologically
confirmation of the DNA results.
● Problem with current attempts to sequence the RhD and RhCE is that they are very
similar gene and standard methods of hybrid capture and amplicon sequencing do not
work
○ Some parts of RhD and RhCE are so similar so sequencing without long enoug
reads can fail to identify which DNA come from RhD and which from RhCE
● The approach we used is very long range PCR
○ It can create gene specific sequences, which circumvents the problem that the
genes look very similar
○ By allowing us to add gene specific tags so we can know which gene it came
from when sequencing
● First step is find a primer that would amplify the Rhd gene DNA
○ Looked at PCR products to determine size and quantity
● Next the goal is to sequence the DNA, using an illumina miseq,
● Take sequencing data dn see which genes they align to
● Last is to examine the sequencing data using a software to determine whether the
sequencing allows us to conclude that the someone has an RhD antigen or if the
sequencing is inconclusive
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926707/
Picture
http://fce-study.netdna-ssl.com/2/images/upload-flashcards/10/70/84/9107084_m.jpg
http://img.medscapestatic.com/pi/meds/ckb/47/9947tn.jpg
5. Presentation
I. Intro
A. What are the consequences of not matching type when giving stem cell
transplants
1. Stem Cell transplants are typically given to leukemia and lymphoma
cancer patients, who have had their immune systems destroyed by
treatment and need the transplants to replenish their immune system
2. GvHD (graft vs. Host Disease)
a) Diseases can be fatal
3. HDFN or HTR for RBC
B. How can DNA help provide more accurate blood typing?
1. For the past 5 yrs HLA (human leukocyte antigens) aka white blood cell
antigens have been typed completely from DNA
2. No longer needs to be done serologically, time consuming process, prone
to human error, expensive lab procedures and reagents
3. However RBC (red blood cell) typing still cannot be confirmed from a DNA
sample but must be tested serologically
C. Our question
1. We chose to look at the RhD and RhCE antigens, to determine whether
we could create a targeted NGS assay to reliably determine the D, C, c,
E, e antigens present on a patient's RBC from a DNA smaple
a) Explain what D anitgen is, determine (+) or (-) blood type
2. These antigens are especially problematic to determine with DNA
sequencing because they are very similar to each other, they can even
containing completely identical exons, and often sequences from one
gene can align to another, for accurate typing it is essential that reads
(sequences) align to the appropriate gene
II. Set Up - ask for Illumina Prep procedure
A. Before we could sequence the RhD and RhCE genes we had to determine which
primers would give us the highest concentration of the genes and the most
accurate “cuts”
1. We focused only on the first 7 exons in both - WHY
B. Performed 12 very long range PCR for each RhD and RhCE with different
primers, and used a gel to determine how much volume was produced, and a
standard to approximate the length of the DNA strands
1. Show image of PCR explain how each well increases in size as it
contains more exons
C. Before putting the the DNA in the Illumina miseq, we tagged the DNA that was
Rhd and that was RhCE, with different tags, so at the end of the process we
know which DNA came from where
1. How does the tagging work???
6. D. The Illumina miseq take about 22 hrs to sequence the gene and then we
download the data into IGV (Integrative Genomics Viewer) to analyze the
success of each of the different primer set combinations
III. Data and Interpretation
A. Show the what the RhD and RhCE look like on one of the most successful trials
and one of the mediocre trials
1. Explain how to tell whether someone has high or low coverage
2. Maybe how to determine their genotype from the one base pair
3. Explain why there might be low coverage
a) Misalignment - graphic from Dr. Lanes Slide
IV. Context and Importance and Future Directions
A. Only 1 of the more than 300 RBC antigens, however it's the most important
antigen after the ABO group
B. When searching for Donors, in the future one sample of spit or cheek swab would
allow a doctor to determine your entire specific blood type,
