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Sample Prep Solutions for Microbiome Research

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An accurate molecular analysis of the microbial constituents of a particular community is contingent upon high-yielding and non-biased nucleic acid extraction methodologies. Only by ensuring that all species and classes of microorganisms present in a sample are effectively lysed during extraction will one be able to reliably assess the composition of that sample. An additional challenge faced in nucleic acid extraction is the presence of persistent, co-purifying polymerase inhibitors endogenous to one’s sample. This presentation will focus on nucleic acid extraction tools developed by MO BIO Laboratories that facilitate accurate non-biased community analysis and eliminate common amplification problems via the depletion of endogenous polymerase inhibitors using our patented Inhibitor Removal Technology.

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Sample Prep Solutions for Microbiome Research

  1. 1. Sample to Insight Sample Prep Solutions for Microbiome Research Eddie Adams, Ph.D. MO BIO Laboratories, a QIAGEN Company Eddie.Adams@qiagen.com
  2. 2. Sample to Insight Legal disclaimer 2 QIAGEN and MO BIO products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor. MO BIO kit manuals are available at www.mobio.com or can be requested from MO BIO Technical Services.
  3. 3. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 3 1 2 3 4 5 6
  4. 4. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 4 1 2 3 4 5 6
  5. 5. Sample to Insight Microbiome: Definition and background 5 What does “microbiome” mean? “The microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that live inside and on the human body.” -NIH, 2012 Microbiota refers to the collection of microbial organisms that inhabits a certain environment Metagenomics is the study of the collective genomes of microorganisms from a sample without cultivation (Lederberg and McCray 2001, The NIH HMP Working Group) Kuczynski et al. Nature Reviews Genetics 13,47-58 (January 2012)
  6. 6. Sample to Insight Human Microbiome Project 6 Microorganisms cluster by body site Cataloguing efforts by the NIH Human Microbiome Project suggest: • ~10,000 organisms live with us • ~ 8 ×106 genes in this “second genome” Identifying microbiota in healthy individuals revealed: • Different body sites have unique communities • Race, age, gender, weight or ethnicity have an effect 1 Hoffmann A.R., et al. “The Microbiome: The Trillionsof MicroorganismsThat Maintain Healthand Cause Disease in Humansand Companion Animals.” Vet Pathol.2015 2 http://commonfund.nih.gov/hmp/index 3 Structure, functionand diversity of the health human microbiome. The HumanMicrobiome Project Consortium. Nature,486, 207 -214 (14 June 2012). doi: 10.1038/nature11234
  7. 7. Sample to Insight Earth Microbiome Project 7 Multidisciplinary effort to survey the microbial composition of diverse environments across the globe: • Aims to process 200,000 samples from these different biomes • Generate a database of microbes and their gene products • Will greatly enhance our understanding of the roles microbes play in ecology • Will expand our understanding of microbial metabolism and gene products • Estimates of bacterial diversity1: • 160 distinct types of bacteria in 1 ml of ocean water • 6,400 – 38,000 types of bacteria in 1 gram of soil These are just estimates for bacteria alone; one still needs to consider viruses, archaea and fungi 1Curtis, T. P.; Sloan, W. T.; Scannell, J. W. (2002). "Estimating prokaryotic diversity and its limits". Proceedings of the National Academy of Sciences 99 (16): 10494–10499.
  8. 8. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 8 1 2 3 4 5 6
  9. 9. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 9 1 2 3 4 5 6
  10. 10. Sample to Insight Molecular technologies for microbial communityanalysis 10 16S rRNA gene sequencing 18S rRNA gene sequencing Total DNA sequencing (shotgun) Bacteria and Archaea Fungus / Yeast Viruses Gene content RNA expression profiling (transcriptomics) Gene expression Metabolite, protein characterization Mass spectroscopy (metabolomics & proteomics) Identify relative frequencies and pathways Microbiome sample Extract DNA Extract RNA Extract proteins & small molecules What organisms are present and what is their relative abundance? What are the functions of the community?
  11. 11. Sample to Insight Sample preparation requirements for successful microbiome studies There are several areas where sample prep inefficiencies can bias a microbiome/metagenomics study: • Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate interactions) • Insufficient cell lysis ◦ Can bias downstreamanalysis toward ‘the easily disrupted’population(s) • Poor nucleic acid quality, extensive shearing • Insufficient inactivation of nucleases/proteases ◦ Unintended destructionof template molecules of interest • Insufficient solubilization of analyte(s) of interest/separation from interacting cellular components • Unintended precipitation of nucleic acids via complexation with matrix-derived metals, bioactive amines • Low-yielding binding interactions with purification matrices • Co-purification of small molecule inhibitors of amplification reactions (e.g., PCR) ◦ Decreases efficiency of amplification or can completely inhibit library prep reactions
  12. 12. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 12 1 2 3 4 5 6
  13. 13. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 13 1 2 3 4 5 6
  14. 14. Sample to Insight About MO BIO Laboratories: • 22 year-old Carlsbad, CA molecular biology company – Privately owned until acquisition by QIAGEN in November 2015 • Current catalog of products will be manufactured in Carlsbad until end of 2016; QIAGEN (Hilden, Germany) will then manufacture all products from 2017 onward • Customers will still be able to order MO BIO products through current website (www.mobio.com) and products will be supported by current MO BIO Technical Support staff • ~50 employees • Products sold in 90+ countries MO BIO’s sample prep solutions
  15. 15. Sample to Insight High Inhibitors Difficult Lysis MO BIO’s sample prep solutions Easy Difficult Lysis Inhibitors Low High Blood, animal tissue & cells Pure microbial cultures Soil microbesLeaf tissue Stool & gut microbes Biofilm FFPE Tissue Food cultures
  16. 16. Sample to Insight MO BIO’s sample prep solutions Cell lysis with homogenous gene/community representation • Mechanical homogenization with tailored lysis buffer formulations (DNA and RNA, DNA or RNA only, protein) Increased purity • Inhibitor Removal Technology® – IRT (“Power” line of kits) Customizable throughput • From single silica spin filters to unique non-fouling ClearMag™ beads in high-throughput,automated applications
  17. 17. Sample to Insight Bead-basedhomogenization for unbiased nucleic acid representation Metal 2.38 mm Ceramic 1.44 & 2.8 mm Glass 0.1 & 0.5 mm Carbide 0.25 mm Garnet 0.15 & 0.70 mm Vortex bead tube adapters Inexpensive means for sample homogenization and lysis (max 24 samples) PowerLyzer24 High-speed homogenizer for hard-to-lyse samples (max 24 samples) TissueLyser II High-speed homogenizer for hard-to-lyse samples (up to 192 samples)
  18. 18. Sample to Insight Sample-derived PCR/RT-PCR inhibitors In the process of breaking open cells to release nucleic acids, amplification inhibitors are also released. Inhibitors include humic/fulvic acids in soil and bile, bilirubin and heme in stool.* *Other examples for additional matrices given in Rådström, P. et al. (2004) Pre-PCR processing: Strategiesto generate PCR-compatible samples. Mol. Biotechnol. 26, 133–46.
  19. 19. Sample to Insight Sample-derived PCR/RT-PCR inhibitors Sample-derived PCR inhibitors can act via several overlapping mechanisms • Sequestration of reaction components/enzyme co-factors ◦ Mg2+ chelation by sample-derived carboxylates • Competitive displacement of reaction co-factors ◦ Ca2+ displacement of Mg2+ • Direct interaction with template nucleic acids ◦ Competitive displacement of enzyme ◦ Steric occlusion of enzyme • Direct interaction with polymerase
  20. 20. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 20 1 2 3 4 5 6
  21. 21. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 21 1 2 3 4 5 6
  22. 22. Sample to Insight Inhibitor Removal Technology® The inhibitor removal chemistry outlined in US 7,459,548 forms the basis for all MO BIO “Power” kits (e.g., PowerSoil, PowerMicrobiome).
  23. 23. Sample to Insight Inhibitor Removal Technology® IRT removes the most challenging inhibitors from lysates prior to nucleic acid purification. Comparison of samples with and without IRT: Inhibitors cause false negatives in qPCR, RT-qPCR and sequencing analysis.
  24. 24. Sample to Insight No IRT IRT Method IRT MethodNo IRT No IRT IRT No IRT No IRT IRT IRT Why contaminationremoval is important
  25. 25. Sample to Insight Low levels of contaminating inhibitors can lead to false negatives / aberrant amplification IRT + + - - + IRT - IRT Samples 260/280 260/230 IRT 1.91 2.03 IRT 1.92 1.99 No IRT 1.87 1.84 No IRT 1.85 1.53
  26. 26. Sample to Insight Projects using MO BIO Kits with Inhibitor Removal Technology®
  27. 27. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 27 1 2 3 4 5 6
  28. 28. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 28 1 2 3 4 5 6
  29. 29. Sample to Insight DNeasy PowerSoil Kit (formerly PowerSoil DNA Isolation Kit)* 29 MO BIO’s most important offeringto the microbiome community *Though developed for soil, this kit is often used for stool, swabs, and a host of other sample matrices. Often used interchangeably with the QIAamp PowerFecal Kit (formerly PowerFecal DNA Isolation Kit).
  30. 30. Sample to Insight In the literature: Frossard, A. et al., (2015) Water regime history drives responses of soil Namib Desert microbial communities to wetting events. Sci. Rep. 5, 12263 • The effects of wetting event frequency and intensity on Namib Desert microbial communities from two soils with different water-regime histories were tested over 36 days • The intensity of the water pulses (i.e. the amount of water added) rather than the frequency of wetting events had greatest effect in shaping bacterial and fungal community structures • In contrast to microbial diversity, microbial activities (enzyme activities) showed very little response to the wetting events and were mainly driven by soil origin. • Conclusion: While microbial community structures might be irreversibly altered by the successive dry and wet cycles, microbial activities are expected to be more resilient, suggesting functional redundancy of the microbial communities
  31. 31. Sample to Insight MagAttract PowerSoil DNA Kit (formerly PowerMag Soil DNA Isolation Kit) • Uses the same chemistry as PowerSoil/PowerFecal kits for lysis & IRT • Magnetic bead purification of DNA enables robotic automation of all steps post-IRT
  32. 32. Sample to Insight In the literature: Quinn, R.A. et al., (2016) From sample to multi-omics conclusions in under 48 hours. mSystems, 1, 2. Applied an integrated omics pipeline for human and environmental samples & reported full analysis of integrated data set w/in 48 hours. • 16S rRNA gene sequencing • Inferred gene function profiles • LC-MS/MS metabolomics Swab samples from skin, feces, oral cavity, fermented foods and household surfaces. Conclusions: • Human samples clustered with corresponding types in American Gut Project data set • Fermented foods produced distinct cluster • Microbial communities of household surfaces derived primarily from fermented foods • Modified and unmodified metabolites derived from fermented foods detected in stool • Multi-omics analysis achieved on time scales similar to classical microbiological culturing
  33. 33. Sample to Insight RNeasy PowerMicrobiome Kit (formerly PowerMicrobiome RNA Isolation Kit ) Microbial RNA from (a) stabilized dog stool and (b) dog stool transported on ice. a b
  34. 34. Sample to Insight In the literature: Reck, M. et al., (2015) Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation. BMC Genomics 2015 16:494 Authors present a workflow for the stabilization of stool microbial cells/nucleic acids and their extraction. Compared 3 commercial kits and one literature methodology • Determined that the PowerMicrobiome kit performed the best with respect to RNA yield and purity. Paired the PowerMicrobiome kit with several commercial stabilization reagents and analyzed extracted mRNA via Illumina HiSeq compared to snap-frozen controls • mRNA transcripts preserved in RNAlater unchanged for up to 6 days at RT but a bias was clearly detected (reduced abundance of Prevotellaceae transcripts & depleted transcripts for COG category “Carbohydrate transport & metabolism” Conclusion: RNAProtect + PowerMicrobiome yielded transcriptomes most similar to snap-frozen controls
  35. 35. Sample to Insight DNeasy PowerBiofilm Kit (formerly PowerBiofilm DNA Isolation Kit)
  36. 36. Sample to Insight In the literature: Abed, R.M. et al., (2014) Diversity, distribution and hydrocarbon biodegradation capabilities of microbial communities in oil-contaminated cyanobacterial mats from a constructed wetland. PLoS ONE 9(12) Constructed wetland treatment systems are devised to exploit natural processes for the cleanup of wastewaters. “Producedwater” = water from underground formations brought to the surface via wells during oil & gas production. • Even after oil & gas separation, produced water is still contaminated w/residual hydrocarbons. • 50 million m3/day of produced water = environmental challenge for energy industry Authors studied cyanobacterial mats in BAUER-commissioned wetland system that treats 95,000 m3 of oil-field production water/day. Conclusions: The wetland mats were able to degrade 53–100% of C12–C30 alkanes after 6 weeks of incubation under aerobic conditions. Oil and ammonia concentrations are the major key players in determining the spatial distribution of the wetland mats’ microbial communities. Mats contribute directly to the removal of hydrocarbons fromoil field wastewaters..
  37. 37. Sample to Insight Sample to Insight: MO BIO sample prep with QIAGEN’s microbial qPCR products 37 Microbiology: From identification to characterization 16S rRNA gene - Conserved region - Variable region Microbial qPCR assays and arrays for identification and profiling use probes and primers against 16srRNA variable region. Content: Largest microbiome portfolio; experimentally verified 580 assays Custom: Select 8 to 384 microbial species for simultaneous detection / profiling Control: Integrated controls ensure reliability of results Sensitivity: Can detect as low as 10 copy numbers; data available
  38. 38. Sample to Insight Overview of QIAGEN’s microbial qPCR products 38 Microbial DNA qPCR Array: Pre-printed assays profile up to 90 different species/genes PCR plates (either 96-well or 384-well) are pre-printed with primers and probes. Each numbered well is a separate assay that tests the same sample. Integrated control assays: • Host assays detect genomic DNA to test sample collection • Pan A/C is a pan- Aspergillus/Candida assay that detects the presence of fungal rRNA • PanB1 and PanB2 detect bacterial 16S rRNA to determine bacterial load in the sample • PPC is a positive PCR control reaction that tests if the PCR reactions failed due to PCR inhibitors in the sample, etc.
  39. 39. Sample to Insight Overview of QIAGEN’s microbial qPCR products 39 Layout of a Microbial DNA qPCR Array: Different arrays have different number of assays and samples
  40. 40. Sample to Insight Overview of QIAGEN’s microbial qPCR products 40 Microbial DNA qPCR Arrays and Assays Profile or identify the presence of microbial DNA(from bacteria, fungi, virus, protist, antibiotic resistance and virulence factors) Identification experiment answers the following question: Are any of these microbes or genes present in the sample? • Must be compared against a known negative sample • Run NTC as one sample • Answers are Yes or No Profiling experiment answers the following question: Have the amounts of any of these microbes or genes changed? • Must be compared against a reference sample • Answers are fold change
  41. 41. Sample to Insight Integration of MO BIO products into the Sample to Insight workflow 41 Sample to Insight : Inhibitor-depleted nucleic acids  Microbial qPCR Assays and Arrays QIAsymphony/QIAcube/QIAcube HT QIAgility Rotor-Gene Q DNA Isolation Assays and Arrays Data Analysis • DNeasy PowerSoil • DNeasy PowerFecal • RNeasy PowerMicrobiome • DNeasy PowerBiofilm DNA • RNeasy PowerBiofilm • MagAttract PowerSoil DNA • DNeasy Microbial DNA • Microbial DNA qPCR Arrays • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • Microbial qPCR Multi-Assay Kits • Custom Microbial DNA qPCR Arrays • GeneGlobe Data Analysis Center Sample Insight
  42. 42. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Microbial NGS (microbiome / pathogen): MO BIO + QIAGEN Tools Sample Disruption Sample Preparation Library construction NGS run Data analysis Validation by PCR • TissueLyserII; TissueLyser LT; TissueRuptor; PowerLyzer 24; Vortex adapters • Pathogen Lysis Tubes • Power Bead Tubes Data Analysis Software • CLC Bio Genomics workbench • Microbial Genome Finishing module Predesigned & custom arrays / assays for verification and focused microbiome analyses • Microbial DNA qPCR Arrays • Microbial DNA qPCR Multi-Assay Kits • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • QIAamp PowerFecal • DNeasy o PowerSoil o PowerWater o PowerBiofilm • RNeasy PowerMicrobiome • AllPrep PowerFecal • Repli-g Single Cell Kit (Limited primary sample material) • QIASeq FXDNA Library Kit for high-complexity metagenome libraries • REPLI-g Single Cell Kit for whole genome amplification from unculturable organisms or limited samples • QIASeq 1-Step Amplicon Kit the fastest solution for targeted metagenomics libraries • QIAGEN Multiplex PCR kits for effective 16s, 18s or ITS amplification with minimal PCR- bias.
  43. 43. Sample to Insight Want to stay up-to-date? Visit www.mobio.com to see our complete kit catalog, find Technical Support FAQs, and read our microbiome blog with great guest writer entries. Stay up to date with developments in microbiome news at the Microbiome Project (http://news.microbiomeproject.com/). All products are now on www.qiagen.com @mobio Follow us: MO BIO Laboratories
  44. 44. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 44 1 2 3 4 5 6
  45. 45. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial community analysis MO BIO’s sample prep solutions Inhibitor Removal Technology® (IRT) MO BIO’s microbiome product offerings Questions 45 1 2 3 4 5 6
  46. 46. Sample to Insight Thank you for coming Any questions? Contact us QIAGEN Telephone: 888-503-3187 Webinar questions: qiawebinars@QIAGEN.com Technical support: techservice-na@qiagen.com Contact us MO BIO Laboratories, Inc. Toll-free in USA: 800-606-6246 Outside USA: 760-929-9911 46

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