QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep
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In this slide deck, learn about the innovative technologies that form the basis of QIAGEN’s portfolio of QIAseq library prep solutions for metagenomics and microbiome sequencing. Whether your research starts from single microbial cells, 16s rRNA PCR amplicons, or gDNA for whole genome analysis, QIAseq technologies offer tips and tricks for capturing the genomic diversity of your samples in the most unbiased, streamlined way possible.
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QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep
- 1. Sample to Insight QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep Jen Fostel, Sr. Global Product Manager 1QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
- 2. Sample to Insight 2 • QIAGEN 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. Legal disclaimer QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016
- 3. Sample to Insight QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 3 QIAseq Sample to Insight solutions QIAseq: QIAGEN’s portfolio of Sample to Insight solutions for Illumina and Ion Torrent sequencers. From liquid biopsy and oncology biomarker discovery to metagenomics and single cell sequencing, our application-specific products provide researchers with comprehensive solutions to deliver the next wave of innovation in the genomics community.
- 4. Sample to Insight Agenda Introduction to microbial and metagenomics NGS 16S/18S rRNA phylogenomic community profiling Whole genome and functional metagenomics NGS from isolated bacterial cells Wrap up and questions QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 4 1 2 3 4 5
- 5. Sample to Insight Agenda Introduction to microbial and metagenomics NGS 16S/18S rRNA phylogenomic community profiling Whole genome and functional metagenomics NGS from isolated bacterial cells Wrap up and questions QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 5 1 2 3 4 5
- 6. Sample to Insight Microbiome and metagenomic terminology QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 • Human cells are outnumbered by a factor of 10 by microbial cells that live in and on the human body. These microbial cells comprise the human microbiome. • Terminology: o Metagenomics is the study of the collection of genomes derived from a specific sample or community o Microbes are microscopic organisms that can be either single or multicellular o Microbiota are the microbes that live in a specific location (e.g., the human body, the gut, soil, etc.) o Microbiome: collective genes of these microscopic co-inhabitants in a system (e.g., human host) 6 Source: Human mMcrobiome Project, NIH http://commonfund.nih.gov/hmp/index
- 7. Sample to Insight Environmental microbiomes and metagenomics Multidisciplinary effort to survey the microbial composition of diverse environments across the globe: • Aims to process 200,000 samples from different biomes and generate a database of microbes and their gene products • Estimates of bacterial diversity: o 160 distinct types of bacteria in 1 ml of ocean water o 6400–38,000 types of bacteria in 1 gram of soil QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 7 Source: Curtis, T.P., Sloan, W.T. and Scannell, J.W. (2002) Estimating prokaryotic diversity and its limits. Proc Natl Acad Sci USA 99, 10494–9. These estimates are for bacteria alone; they do not include viruses, archaea or fungi
- 8. Sample to Insight Cataloging microbial communities QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 Current Methods for Microbial Analysis • Culture • Gene cloning (Pan 16S rRNA) and Sanger sequencing • Microarray • Next-generation sequencing o 16S rRNA sequencing o Whole genome sequencing • MALDI • qPCR – Target dependent o 16S rRNA gene o Other relevant gene (antibiotic resistance gene, virulence factor gene) 8
- 9. Sample to Insight Excellent correlation between qPCR and NGS profiling QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 Profiles of vaginal flora by qPCR and whole genome sequencing 9
- 10. Sample to Insight Choosing technology for microbial analysis and metagenomics Speed and specificity vs. discovery at scale QIAseq NGS library prep QIAGEN microbial qPCR assays • Highest throughput • Compatible with both 16S rRNA-seq and genome-wide discovery • Specific, easy-to-set-up, off- the-shelf assays • Fastest assay and analysis: under 3 hours QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 10
- 11. Sample to Insight Agenda Introduction to microbial and metagenomics NGS 16S/18S rRNA phylogenomic community profiling Whole genome and functional metagenomics NGS from isolated bacterial cells Wrap up and questions QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 11 3 4 5 2 1
- 12. Sample to Insight • 16S rRNA gene as a phylogenetic marker for bacterial ID • Sequence similarity = 95% genus level, 97% species level, 99% strain level • Classification from the variable sequences QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 12 16S rRNA community profiling
- 13. Sample to Insight Example data: findings from the NIH Human Microbiome Project Microorganisms cluster by body site Cataloguing efforts by the NIH Human Microbiome Project suggest: • Different body sites have unique communities • Race, age, gender, weight and ethnicity all have effects on microbiome populations QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 13 Sources: Rodrigues Hoffman, A., Proctor, L.M., Surette, M.G., Suchodolski, J.S. (2016) The microbiome: the trillions of microorganisms that maintain health and disease in humans and companion animals. Vet Pathol 53, 10–21. Human Microbiome Project, NIH https://commonfund.nih.gov/hmp/index Human Microbiome Project Consortium. (2012) Structure, function and diversity of the healthy human microbiome. Nature 486: 207–14.
- 14. Sample to Insight Microbial qPCR assays and multiplex PCRs for identification and profiling use probes and primers against 16S rRNA variable regions. 16S rRNA PCR Library prep Cleanup Library amplification Sequencing QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 14 16S rRNA community profiling
- 15. Sample to Insight QIAseq 1-Step Amplicon QIAseq NGS library prep kits SequencingDNA/RNA extraction 30-minute, room temperature library prep for PCR products 16S rRNA community profiling QIAseq FX DNA Library Kit All-enzymatic workflow without Covaris shearing or GC bias WGS or long-range PCR/cDNA-seq QIAseq FX Single Cell DNA Library Kit From single isolated cells to libraries in under 4 hours Bacterial single cell WGS QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 15 QIAseq 1-Step Amplicon for the simplest amplicon library prep
- 16. Sample to Insight QIAGEN recomends: QIAseq 1-Step Amplicon Library Kit 30-minute single-tube, room-temperature library prep for amplicon-targeted resequencing • Proprietary single-tube approach: only one pipetting step • Fastest possible library prep: 30 minutes, room-temperature incubation Library amplification (optional) (45 min) Single-tube room-temperature library prep (30 min) 16S rRNA PCR product (1–500 ng) QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 16
- 17. Sample to Insight QIAGEN solution for 16S rRNA: dramatically faster library prep QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 17
- 18. Sample to Insight QIAseq 1-Step Amplicon Library Kit: contents and advantages Kits contain: • Enzymes and buffers for 1-step library preparation • Single-use, 96-plex Illumina adapters (96-reaction format only) • HiFi polymerase and buffer for library amplification (optional) What’s not included: • Agencourt AMPure XP beads for reaction cleanup • qPCR reagents for library quantification: recommended for accurate flow cell loading, especially for PCR-free workflows. Fast and simple library prep Replace 2–3 hours of sequential steps with a single 30- minute reaction Room temperature library prep now completely on the benchtop, perfect for automation QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 18
- 19. Sample to Insight Agenda Introduction to microbial and metagenomics NGS 16S/18S rRNA phylogenomic community profiling Whole genome and functional metagenomics NGS from isolated bacterial cells Wrap up and questions QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 19 2 4 5 3 1
- 20. Sample to Insight NGS whole genome and functional metagenomics Complexity and function of genomic content – function of microbiome enables individual survival • Genetic content development in each organism for its own survival in a specific environment • Metabolism tuned to local nutrient sources • Virulence factors for stable colonization • Antibiotic resistance genes to metabolize toxins QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 20 Image source: Kyoto Encyclopedia of Genes and Genomes (KEGG) http://www.kegg.jp/kegg/
- 21. Sample to Insight Example application: antibiotic resistance detection Antibiotic resistance genes in our food supply? • One potential source of acquiring antibiotic resistance genes is through the food supply. • Both livestock and feed may acquire antibiotic resistant bacteria through different mechanisms. • Food can be exposed to antibiotic resistant bacteria through fertilizer originating from wastewater treatment plants. This exposure, in addition to increasing administration of antibiotics to livestock, can lead to food becoming a potential source of antibiotic- resistant genes. • Consequently, a horizontal gene transfer to pathogenic enteropathogens may result – leading to drug resistance in humans. This dynamic highlights the importance of surveillance and prevention of antibiotic- resistant genes in food. QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 21
- 22. Sample to Insight Example application: QIAseq FX for urban metagenomics QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 22
- 23. Sample to Insight Challenge in whole genome metagenomics: DNA fragmentation Mechanical shearing Tagmentation Fragment DNA Add adapters Amplify and QC library Typical WGS library prep process: • Higher costs • Harder to scale up • Labor-intensive • Strong GC bias • Inflexible input requirement • Inflexible fragment size QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 23
- 24. Sample to Insight Why GC bias makes a difference in metagenomic studies GC content varies widely within and between microbial genomes E. coli B. pertussis F. nucleatum (low GC) • Tagmentation cleaves low GC sequences preferentially • Sequence preference strongly biases species/region detection Tagmentation fragment size: 1 ng DNA QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 24 Source: Inferring horizontal gene transfer, Wikipedia https://en.wikipedia.org/wiki/Inferring_horizontal_gene_transfer
- 25. Sample to Insight QIAseq 1-Step Amplicon QIAseq Library Prep Kit for NGS SequencingDNA/RNA extraction 30-minute, room temperature library prep for PCR products 16S rRNA community profiling QIAseq FX DNA Library Kit All-enzymatic workflow without Covaris shearing or GC bias WGS or long-range PCR/cDNA-seq QIAseq FX Single Cell DNA Library Kit From single isolated cells to libraries in under 4 hours Bacterial single cell WGS QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 25 QIAseq FX for whole genome and functional metagenomics
- 26. Sample to Insight QIAGEN solution: all-enzymatic nuclease-based library prep FX chemistry for random, enzymatic DNA fragmentation included in QIAseq FX Kits: adapter ligation (45 min) QIAGEN HiFi library amp (45 min – optional) Single-tube FX reaction (50–60 min) Purified gDNA 1 ng – 1 µg Ready-to-sequence library QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 26
- 27. Sample to Insight Mechanical-quality fragmentation from an enzymatic workflow Sample-to-sample fragmentation reproducibility 0 200 400 600 5 min 10 min Averagefragmentsize (bp) Fragmentation Time Frag. #1 Frag. #2 Frag. #3 Frag. #4 Customizable fragment size: 0 200 400 600 5 min 10 minAveragefragmentsize (bp) Fragmentation time Input DNA species Bacterial Mix Human 250 bp 450 bp 1000 bp QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 27
- 28. Sample to Insight QIAseq FX exhibits less GC bias than comparable methods 0 0.5 1 1.5 2 0 20 40 60 80 100 Normalizedcoverage GC% over 100 bp regions Genome coverage , 100 ng input QIAGEN QIAseq FX NEB enzymatic fragmentation Covaris + standard library prep (Tagmentation not possible at 100 ng input) 0 0.5 1 1.5 2 0 20 40 60 80 100 GC% over 100 bp regions Genome coverage, 1 ng input QIAGEN QIAseq FX NEB enzymatic fragmentation Covaris + standard library prep Tagmentation QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 28
- 29. Sample to Insight Superior genomic coverage from all-enzymatic library prep 0 0.02 0.04 0.06 0.08 0.1 0.12 0 20 40 60 80 100 Fractionoftargetgenome Coverage depth (x) Genomic coverage distribution QIAseq FX (100 ng) NEB enzymatic fragmentation (100 ng) Covaris + library prep (100 ng) Tagmentation (1 ng) 0 0.5 1 1.5 2 0 20 40 60 80 100 Normalizedcoverage GC% over 100 bp regions Coverage by GC% QIAseq FX (50 ng PCR-free) QIAseq FX (100 ng PCR-free) Covaris + library prep (100 ng) with PCR QIAseq FX (100 ng) with PCR*Supplier I does not offer a kit for tagmentation from 100 ng DNA QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 29
- 30. Sample to Insight Case study: QIAseq FX DNA Library from GC-rich gDNA Paired-end mapping read track Bacteria: Bordetella pertussis, ~67% GC Input gDNA amount: 1 ng Library construction: QIAseq FX Library size (insert + adaptor): 499 bp Library amplification: 10 cycles Data analysis: CLC Genomics Workbench QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 30
- 31. Sample to Insight QIAGEN poster: Phillip Widman at European Microbiome Congress Cattle fecal samples: 5 affected animals 5 unaffected animals Johne’s Disease, or paratuberculosis: • Gastrointestinal disease of ruminants • Mycobacterium avium ss paratuberculosis • Symptoms: rapid weight loss, diarrhea • Diagnosis: PCR, culture AllPrep PowerFecal DNA Isolation Kit DNA 16S rRNA amplification QIAGEN QuantiTect Probe PCR Kit MiSeq; 500 bp paired-end Analysis: CLC Microbial Genomics Module Whole genome metagenomics QIAseq FX DNA Library Kit MiSeq; 500 bp paired-end QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 31
- 32. Sample to Insight Agenda Introduction to microbial and metagenomics NGS 16S/18S rRNA phylogenomic community profiling Whole genome and functional metagenomics NGS from isolated bacterial cells Wrap up and questions QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 32 3 5 1 2 4
- 33. Sample to Insight Challenge: extremely low DNA content in isolated bacterial cells Standard NGS library prep input: 100–1000 ng Bacterium Mammalian cell 200 µl Blood 1 µg 1 ng 1 pg 1 fg AverageDNAcontent Many “standard” NGS library prep methods still require 100 ng or more input DNA QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 33
- 34. Sample to Insight QIAseq 1-Step Amplicon QIAseq Library Prep Kit for NGS SequencingDNA/RNA extraction 30-minute, room temperature library prep for PCR products 16S rRNA community profiling QIAseq FX DNA Library Kit All-enzymatic workflow without Covaris shearing or GC bias WGS or long-range PCR/cDNA-seq QIAseq FX Single Cell DNA Library Kit From single isolated cells to libraries in under 4 hours Bacterial single cell WGS QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 34 QIAseq FX Single Cell kits for NGS from isolated microbial cells
- 35. Sample to Insight QIAGEN Solution: use MDA, not PCR, for whole genome amp Alkaline denaturation Hexamer random primers Phi29 polymerase strand displacement (30°C) • Modified high-fidelity Phi29 polymerase (SensiPhi) o High enzyme processivity – molecular weight product (10–100 kb) o High-fidelity proofreading activity – 1000-fold higher accuracy than that of Taq • Extremely high yields (micrograms) from a single cell without PCR REPLI-g: multiple displacement amplification in QIAseq Single Cell library prep kits Displaced strand becomes a template for replication QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 35
- 36. Sample to Insight Multiple displacement amplification (MDA) by QIAGEN QIAGEN’s REPLI-g MDA method • Random primers are extended at 30°C as the polymerase moves along the gDNA or cDNA strand – displacing the complementary strand while becoming a template, itself, for replication. In contrast to PCR amplification, MDA: • Does not require thermal cycling, which can bias products against high GC sequences. • Has fewer template-binding events but has very long fragments that effectively replicate the genome. QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 36
- 37. Sample to Insight MDA amplifies through gDNA (or cDNA) secondary structures • Denatured gDNA has a complex secondary structure • Consists of regions of ssDNA and dsDNA that can form complicated hairpins and loops QIAGEN’s Phi29-based SensiPhi enzyme handles complex DNA structures to generate extremely long amplicons QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 37
- 38. Sample to Insight Excellent amplification uniformity across wide GC content range Advantages of coverage uniformity: • Better de novo genome assembly • Wide species applicability • Lower total read number required; higher multiplexing • Advantageous for low-pass sequencing strategy 1 pg DH10B DNA, amplified with either REPLI-g Single Cell Kit or by MALBAC; sequenced on MiSeq Illumina (V2, 2 x 150 nt) QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 38
- 39. Sample to Insight QIAseq FX Single Cell DNA Library Kit: Contents Kits contain: • Cell lysis reagents • Enzymes and buffers for whole genome amplification • Enzymatic DNA fragmentation • Single-step NGS library preparation • Single-use, disposable Illumina adapters in 96-well format • Multiple reagent aliquots to reduce contamination risk and freeze-thaw cycles What’s not included: • Agencourt AMPure XP beads for library purification • PCR reagents for library amplification: not needed as the entire workflow is PCR-free • qPCR reagents for library quantification: recommended for accurate flow cell loading QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 39
- 40. Sample to Insight QIAseq FX Single Cell DNA Library Kit: Workflow Cell lysis 15 min WGA 2 h FX library preparation 70 min Purification 20 min Illumina sequencing 3 h 45 min with ~40 min hands-on time Cell lysis • From single eukaryotic or bacterial cells, or from small amounts (pg – ng) of intact gDNA • Starting with 4 µl cell material in PBS (included) Whole genome amplification • Amplified gDNA can be used directly or frozen until needed • Excess amplified gDNA can used for PCR followup studies • Precise quantification of amplified DNA not needed before library prep NGS library preparation • All-enzymatic workflow requires only a standard thermocycler • Convenient 96-plex plate-format adapters • Completely PCR-free workflow QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 40
- 41. Sample to Insight Agenda Introduction to microbial and metagenomics NGS 16S/18S rRNA phylogenomic community profiling Whole genome and functional metagenomics NGS from isolated bacterial cells Wrap up and questions QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 41 3 1 2 5 4
- 42. Sample to Insight QIAseq 1-Step Amplicon QIAseq Library Prep Kit for NGS SequencingDNA/RNA extraction 30-minute, room temperature library prep for PCR products 16S rRNA community profiling QIAseq FX DNA Library Kit All-enzymatic workflow without Covaris shearing or GC bias WGS or long-range PCR/cDNA-seq QIAseq FX Single Cell DNA Library Kit From single isolated cells to libraries in under 4 hours Bacterial single cell WGS QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 42 The QIAseq portfolio for Metagenomics and Microbiome (Illumina)
- 43. Sample to Insight QIAGEN’s microbial analysis DNA isolation and qPCR products Sample to Insight: Microbial DNA qPCR Assays and Arrays QIAsymphony/QIAcube/QIAcube HT QIAgility Rotor-Gene Q DNA isolation Assays and arrays Data analysis • AllPrep PowerFecal DNA Isolation Kit • QIAamp DNA Microbiome Kit • mericon Bacteria Kit • QIAamp UCP Pathogen Mini Kit • QIAamp DNA Stool Mini Kit • QIAamp UCP Pathogen Blood Kit • QIAamp DNA Mini Kit • MagAttract HMW DNA Kit • QIAseq portfolio of library prep kits • Microbial DNA qPCR Arrays • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • MicrobialDNA qPCR Multi-Assay Kits • Custom Microbial DNA qPCR Arrays • GeneGlobe Data Analysis Center • QIAGEN Microbial Genomics Pro Suite (NGS) Sample Insight QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 43
- 44. Sample to Insight QIAseq ligation-based library prep kit contents End repair and a-addition • End-polishing enzyme • End-polishing buffer 10x Adapter ligation • Ultralow Input ligase • Ultralow Input ligase buffer 4x HiFi PCR Master Mix 96-plex adapter oligos (96 reaction size) Nuclease-free water Illumina library amp PCR primer QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 44
- 45. Sample to Insight 96-plex adapter barcode design (equivalent to TruSeq HT) QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 45
- 46. Sample to Insight 46 Questions? Thank you for attending! All our solutions from Sample to Insight on: QIAGEN.com Contact QIAGEN Technical Service Call: 1-800-362-7737 or 1-800-426-8157 for US Call: +49 2103-29-12400 EU Email: qiaseq.ngs@qiagen.com techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIAwebinars@QIAGEN.com QIAseq Technologies for Metagenomics and Microbiome NGS Library Prep, 12.10.2016 www.qiagen.com/us/products/ngs/ngs-life-sciences/metagenomics/
Notes de l'éditeur
- For those of you who are not already involved in microbiome research, I’d like to start out by providing a few motivating examples of why this application is so powerful, as well as a brief overview of some nomenclature that you may hear throughout this webinar and elsewhere in microbiome research. Describe human microbiome figure. Metagenomics: this is the study of the collection of genomes from a specific sample or community Microbes: these are microscopic organisms that are either single or multi-cellular Microbiota: all of the microbes that live in a specific location, this can be the human body, the gut, soil samples. Microbiome: collective genes of these microscopic co-inhabitants in a system, such as a human host. Sometimes you will see this used in the same way that microbiota is used, but in other cases and in our case as well, we will use it in this way when we are talking about the genes of this microbial community.
- Culture – gold-standard method; it has been traditionally used a lot in research, and it is the most cost-effective method to use. Gene cloning (Pan 16S rRNA) and sanger sequencing - Microarray – used a lot to detect the microbial species Next generation sequencing 16S rRNA sequencing – detection and identification and also discovery purposes Whole genome sequencing – and this is used to find novel microbial sequences MALDI – mass spectrometry method qPCR – this is target-dependent and based on the 16s rRNA gene, or any other relevant gene
- These figures show validation data from a bacterial vaginosis study analyzing the same samples using qPCR as well as whole genome NGS. As you can see, correlation between the two methodologies is generally excellent, leaving it up to you, the researcher, to identify the right technology for your research. NOTES: The performance of the Microbial qPCR array was compared to results generated from Next Generation Sequencing (NGS) on four different vaginal swab samples. For NGS, whole genome sequencing was performed and relative abundance of each organism was determined. For the Microbial DNA qPCR Array, 500 ng of genomic DNA was loaded onto a Vaginal Flora Microbial DNA qPCR array. After the PCR run, raw Ct values were compared to the relative abundance as determined by NGS. Lower Ct values indicate presence of higher abundant organisms and higher Ct values indicate the presence of lower abundant organisms. The results show that identification of bacterial species between the two platforms were in high concordance.
- As compared to traditional culturing methods, both PCR-based and NGS analysis of microbial communities allow direct analysis using the nucleic acids present in a sample. QIAGEN offers both approaches – off the shelf, qPCR based assays for fast, specific easy-to-set-up routine testing, as well as products for NGS detection that allow both increased project scale as well as the flexibility to go beyond 16S or targeted approaches to genome wide discovery and functional metagenomics. In this talk, I’ll be specifically presenting QIAGEN’s QIAseq technologies to address 3 NGS-based microbiome and metagenomic approaches: 16S community profiling, whole genome sequencing from bulk DNA samples, and finally sequencing from isolated, single bacterial cells.
- The simplest way to determine the microbes present in any environmental community is to identify reporter genes high in species-specific information and focus a DNA-based assay there. This approach allows identification of species in the community without needing to be able to culture and identify each microbe in the lab. The 16s ribosomal RNA gene is a well-established target for powerful for bacterial community profiling. Due to its pattern of conserved regions (which are excellent for primer design) as well as multiple variable regions, as a single 1500bp locus has the power to make phylogenetic determinations in 95% of genus level comparisons, 97% of species comparisons, and 99% of strain level comparisons. This particular 16s rRNA region includes conserved and variable regions. The variable regions are going to be what provides the information on the genus, species, and strain level. So this is important to create the primers/probes against this specific region, or you can sequence this to compare to the reference sequence. Sequences and primer design recommendations are well-established in the literature, including the GreenGenes taxonomy used at QIAGEN to design our microbial qPCR assays. In most cases, primer designs established for qPCR can be transitioned to NGS detection for a higher throughput workflow.
- One example of the type of analysis possible with successful community profiling data is this body site specific principal component analysis from the NIH human microbiome project. By plotting each microbial species against axes with high separational power, we can see that there are communities of microbes with common features or functions that typically inhabit various body sites even in healthy individuals. Establishing this kind of baseline level understanding of microbial communities also serves as an important first step for diagnosing and understanding the microbiome’s effect on various disease states. Extra: The interest in the microbiome also went up when NIH began their human microbiome project. The Human Microbiome Project (HMP) is also separated into 2 stages. Stage 1 (2008-2013) goal was to “enable the comprehensive characterization of the human microbiome and analysis of its role in human health and disease.” They screened 242 healthy individuals to see if they could discern what a baseline “healthy” microbiome looked like at different body sites. What they found in this study was that there are about 10,000 organisms living on and inside of us, and about 8 million genes from this second genome.
- So how is a traditionally PCR-based assay typically converted to NGS? Most short-read NGS platforms – including Illumina, Ion Torrent, and the QIAGEN gene reader – are ideal for sequencing the products of PCR or primer extension based targeted enrichment. The challenge lies in selecting the NGS library prep products most appropriate for your application.
- All short-read sequencing technologies, including Illumina and Ion Torrent, require high molecular weight genomic DNA to be fragmented into short, typically 200-600 bp pieces prior to library prep. This step is show in the middle of the workflow scheme at the bottom of this slide. As I mentioned, the two market-leading solutions to this fragmentation problem are: First, Covaris: an acoustic shearing technology that requires the purchase of (and lab space for) highly specialized equipment and expensive single-use glass tubes; and Second, Nextera: an all-enzymatic method in which DNA is fragmented and adapters are added at the same time using a transposase or “tagmentation” reaction.
- Low sequence bias also generates more even, predictable genomic coverage. The single, narrow peak for QIAseq FX in the plot on the left shows that the majority of genomic targets have very similar total coverage depth – meaning you don’t need to do a lot of additional sequencing to bring low-coverage targets up into an interpretable range. In contrast, the peak for Nextera (in red) tends to be broader, with a lower average coverage depth generated by the same amount of sequencing. Likewise, the bimodal distribution for NEB Fragmentase (in green) shows that while about half of targets are well-covered, a significant peak near zero contains sequences that are poorly covered or not covered at all. On the right, we show duplication rate, or the rate at which sequence reads are discarded during analysis because they are exact matches for a previous read. Duplication rate tells you what fraction of a dataset was derived from PCR copies created during sample processing rather than new genomic diversity present in the original sample DNA. Libraries with low complexity will have a higher duplication rate, while high quality libraries have sufficient genomic diversity to cluster the flowcell with nearly 100% unique sequences. At 1 nanogram of input, QIAseq FX (in blue, second from the right) performs comparably to Covaris, with sub-1% duplication. This is in contrast to the NEB and Nextera methods tested, which each show significant fractions of the data generated coming from PCR duplicate molecules. At 100ng input (blue, far right), QIAseq FX duplication rates are even lower, approaching the level typically seen from PCR-free workflows.