Semi Automated Low-throughput Workflow for Microbial Analyses of Human Stool
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The document describes a semi-automated workflow for microbial analyses of human stool using Qiagen technologies, which includes DNA purification, library preparation, and bioinformatics. It emphasizes the significance of microbiome studies and the impact of microbial communities on human health, detailing various methodologies for high-quality nucleic acid extraction and analysis. The conclusion highlights the efficient integration of analytics for research continuity in microbial genomics, aimed at facilitating discoveries in microbial diversity and function.
Semi Automated Low-throughput Workflow for Microbial Analyses of Human Stool
- 1. Sample to Insight Semi-automated low-throughput workflow for microbial analyses of human stool Patrick Smith PhD, R&D Scientist, QIAGEN GmbH, Patrick.Smith@qiagen.com
- 2. Sample to Insight Overview Introduction ◦ Microbiome ◦ Inhibitor Removal Technology (IRT) Semi-automated DNA purification ◦ QIAamp® PowerFecal® Kit ◦ QIAcube® automation Library preparation ◦ QIAseq™ 1-Step Amplicon Library Kit (16S) ◦ QIAseq FX DNA Library Kit (WGS) Bioinformatics – CLC Genomics Workbench ◦ CLC Microbial Genomics Module 2 1 2 3 4
- 3. Sample to Insight Overview Introduction ◦ Microbiome ◦ Inhibitor Removal Technology (IRT) Semi-automated DNA purification QIAamp PowerFecal Kit QIAcube automation Library preparation QIAseq 1-Step Amplicon Library Kit (16S) QIAseq FX DNA Library Kit (WGS) Bioinformatics – CLC Genomics Workbench CLC Microbial Genomics Module 3 1 2 3 4
- 4. Sample to Insight The human super-organism 4 Cataloging efforts by the NIH Human Microbiome Project (1) suggest: • ~10,000 organisms live with us • Bacterial genes outnumber our own 150:1 Identifying microbiota in healthy individuals revealed: • Different body sites have unique communities • Race, age, gender, weight or ethnicity have an effect 1. The Human Microbiome Project Consortium. (2012) Nature. 486, 207.
- 5. Sample to Insight Association of human microbiota and disease If the host/microbiota relationship is disrupted, gut microbiota can contribute to disease • Gut ◦ Intestinal infections ◦ Obesity ◦ Inflammatory bowel disease • Airway ◦ Pneumonia and other respiratory infections ◦ Chronic obstructive pulmonary disease ◦ Cystic fibrosis • Urogenital ◦ Bacterial vaginosis ◦ Urinary tract infections ◦ Sexually transmitted diseases • Blood ◦ Sepsis/bloodstream infections • Oral ◦ Periodontitis ◦ Gingivitis 5
- 6. Sample to Insight Molecular technologies for microbial community analysis 6 16S 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 and proteomics) Identify relative frequencies and pathways Microbiome sample Extract DNA Extract RNA Extract proteins and small molecules What organisms are present and what is their relative abundance? What are the functions of the community?
- 7. Sample to Insight Sample preparation for successful microbiome studies Several areas where sample prep inefficiencies can bias a microbiome/metagenomics study: • Co-purification of small molecule inhibitors of amplification reactions (e.g., PCR) ◦ Decreases efficiency of amplification or can inhibit library prep reactions • Insufficient cell lysis ◦ Biases downstream analysis toward ‘the easily disrupted’ population(s) • Reproducible purification of high-quality nucleic acids • Poor nucleic acid quality, extensive shearing • 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 • Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate interactions) 7
- 8. 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 complex polysaccharides, bile, bilirubin and heme in stool* Other examples for additional matrices given in Rådström, P. et al. (2004) Mol. Biotechnol. 26, 133. 8
- 9. Sample to Insight Effect of contaminating inhibitors Low levels of contaminating inhibitors can lead to false negatives/aberrant amplification IRT + + − − + IRT −IRT Low levels of inhibitors can also inhibit other enzymatic reactions commonly found in WGS library prep kits Samples A260/A280 A260/A230 IRT 1.91 2.03 IRT 1.92 1.99 No IRT 1.87 1.84 No IRT 1.85 1.53 9
- 10. Sample to Insight Overview Introduction ◦ Microbiome ◦ Inhibitor Removal Technology (IRT) Semi-automated DNA purification ◦ QIAamp PowerFecal Kit ◦ QIAcube automation Library preparation ◦ QIAseq 1-Step Amplicon Library Kit (16S) ◦ QIAseq FX DNA Library Kit (WGS) Bioinformatics – CLC Genomics Workbench ◦ CLC Microbial Genomics Module 10 2 1 3 4
- 11. Sample to Insight Microbial DNA isolation Sample collection DNA purification using the QIAamp PowerFecal Kit 11 Microbial cell lysis Inhibitor removal Library prep • Mechanical lysis • Patented Inhibitor Removal Technology • QIAamp PowerFecal Kit • All-in-one protocol for sample preparation and removal of inhibitors • Sample material inherently rich in inhibitors: human stool, mammalian stool (mouse, cow, horse), bird stool, environmental samples (DNeasy® PowerSoil® Kit) • Downstream applications: ◦ Whole metagenome shotgun sequencing ◦ 16S rRNA gene sequencing ◦ qPCR
- 12. Sample to Insight QIAcube: Purification QIAcube in NGS, Hilden, November 2015 12 Sample NGS run InsightSample prep on QIAcube • Low-throughput (12 samples per run) automated nucleic acid purification • DNA, RNA and protein purification • No change from manual spin-column procedure • Benchtop, plug and play • Elimination of manual processing steps • Standardized protocols ensure control • 130 standardized protocols • Customizable protocols also available
- 13. Sample to Insight Microbial DNA isolation Sample collection DNA purification using the QIAamp PowerFecal Kit 13 Microbial cell lysis Inhibitor removal Library prep Performed manually Performed by QIAcube • Following mechanical lysis the supernatant is loaded directly into the QIAcube • QIAcube performs all inhibitor removal, DNA binding, wash and elution steps • High-quality DNA is purified (hands-free) in ~45–60 min and can be used immediately in downstream applications
- 14. Sample to Insight Semi-automated DNA purification: QIAamp PowerFecal Kit (1) 14 • Human stool, N=10 • Aged samples: Female (77), female (78), female (74), male (77), male (65) • Young samples: Female (28), female (49), male (28), male (28), male (37) • Additional metadata collected: Ethnicity, diet Microbial DNA isolation Sample collection Microbial cell lysis Inhibitor removal Library prep Performed manually Performed by QIAcube
- 15. Sample to Insight Semi-automated DNA purification: QIAamp PowerFecal Kit (2) 15 Purification of high-quality DNA • 200 mg human stool lysed in PowerBead tubes for 10 minutes using a vortex adapter • Supernatant was then placed in QIAcube for DNA purification • Agarose gel electrophoresis shows purification of high-molecular-weight genomic DNA with minimal degradation • For each sample, several micrograms of DNA was purified, as measured by a fluorescence- based assay 0 5 10 15 Old Young DNA(µg)
- 16. Sample to Insight Semi-automated DNA purification: QIAamp PowerFecal Kit (3) 16 Purification of high-quality DNA • Purified DNA had high A260/A280 and A260/A230 ratios: ◦ Average A260/A280 = 1.8 ◦ Average A260/A230 = 1.7 • PCR inhibition was measured using QIAGEN QuantiFast® Pathogen PCR + IC Kit • 5 µl DNA eluate was added to each reaction • If inhibitors are present, change in CT value is typically between 3 and 15 CTs • DNA is ready for immediate use in downstream applications
- 17. Sample to Insight Overview Introduction ◦ Microbiome ◦ Inhibitor Removal Technology (IRT) Semi-automated DNA purification ◦ QIAamp PowerFecal Kit ◦ QIAcube automation Library preparation ◦ QIAseq 1-Step Amplicon Library Kit (16S) ◦ QIAseq FX DNA Library Kit (WGS) Bioinformatics – CLC Genomics Workbench ◦ CLC Microbial Genomics Module 17 1 4 3 2
- 18. Sample to Insight Whole genome sequencing library prep Methods for microbiome analyses 16s rRNA gene sequencing for community composition Amplicon sequencing Data analysis Mixed sample DNA purification − host and microbes 16S rDNA amplification Community composition Diversity within and between samples Ashelford, K.E., et al. (2005). Appl. Environ. Microbiol. 71, 7724. 18
- 19. Sample to Insight 16S rRNA gene library prep (1) 19 • 50 ng inhibitor-free DNA in 16s rDNA PCR • Modified 515f PCR primer and 806r PCR primer • Addition of adapters with indices using the QIAseq 1-Step Amplicon Kit • 2 x 250 bp on Illumina® MiSeq® Forward primer 5' Reverse primer Adapter Adapter V4 BC 3' BC 5'3' About 350 bp Caporaso, J. G., et al. (2012). ISME J. 6, 1621.
- 20. Sample to Insight 16S rRNA gene library prep (2) 20 • 16S PCR amplicons – 22 cycles • Inhibitor removal from DNA allows efficient amplification/yield of V4 16S region Young samplesOld samples Blanks -500 -300 -1000 bp
- 21. Sample to Insight 16S rRNA gene library prep (3) 21 • Addition of adaptors with indices using QIAseq 1-Step Amplicon Kit • 500 ng amplicon as input, all libraries >10 nM final concentration
- 22. Sample to Insight Overview Introduction ◦ Microbiome ◦ Inhibitor Removal Technology (IRT) Semi-automated DNA purification ◦ QIAamp PowerFecal Kit ◦ QIAcube automation Library preparation ◦ QIAseq 1-Step Amplicon Library Kit (16S) ◦ QIAseq FX DNA Library Kit (WGS) Bioinformatics – CLC Genomics Workbench ◦ CLC Microbial Genomics Module 22 1 4 3 2
- 23. Sample to Insight Whole genome sequencing library prep (1) Pure gDNA (1 ng− 1 μg) Single- tube FX reaction (60 min) Adapter ligation (45 min) Accepts wide range of input DNA Easily customizable fragment sizes Adapter ligation in a single step, with dual indices Fully compatible with Illumina sequencing Fragment peak size 250 bp 350 bp 450 bp 550 bp Fragmentation time (min) at 32°C 10 ng input DNA 24 16 14 10 100 ng input DNA 16 10 8 6 1000 ng input DNA 14 8 6 4 Guideline for choosing the initial fragmentation time Purification sequencing 23
- 24. Sample to Insight Whole genome sequencing library prep (2) 24 Purified librariesStandards 500- 300- 1000- bp • 500 ng input DNA – target fragment size = 350 bp • Agarose gel shows amplicons from qPCR assay using the QIAseq Library Quant Assay Kit • qPCR primers bind to adapters Pure gDNA (1 ng− 1 μg) Single- tube FX reaction (60 min) Adapter ligation (45 min) Accepts wide range of input DNA Easily customizable fragment sizes Adapter ligation in a single step, with dual indices Fully compatible with Illumina sequencing Purification sequencing
- 25. Sample to Insight 16S and WGS sequencing statistics 25 • 16s: 2 x 250 bp with Illumina MiSeq ◦ Generated >22 million paired-end reads ◦ ~ 90% reads passed quality filter, %>Q30 85 ◦ ~ 88% of reads were assigned to an index ◦ Of reads identified, 9 of 10 samples were between 9 and 11% of total reads • WGS: 2 x 250b p with illumina MiSeq ◦ Generated >27 million paired end reads ◦ 97% reads passed quality filter, %>Q30 88 ◦ 88% of reads were assigned to a dual index ◦ Of reads identified, 9 of 10 samples were between 8 and 10% of total reads • DNA preparation through library prep and sequencing worked well!
- 26. Sample to Insight Overview Introduction ◦ Microbiome ◦ Inhibitor Removal Technology (IRT) Semi-automated DNA purification ◦ QIAamp PowerFecal Kit ◦ QIAcube automation Library preparation ◦ QIAseq 1-Step Amplicon Library Kit (16S) ◦ QIAseq FX DNA Library Kit (WGS) Bioinformatics – CLC Genomics Workbench ◦ CLC Microbial Genomics Module 26 1 2 3 4
- 27. Sample to Insight QIAGEN Microbial Genomics Pro Suite (1) 27 From data to discovery • Integrated analytics deliver research continuity ◦ All analytics for microbial genomics and metagenomics fully integrated ◦ Fully scalable with data and sample-metadata management included • Focus on what matters ◦ Streamlined workflow allows users to focus on interpretation of results • High-performance algorithms ◦ Designed to save time and compute resources ◦ Accessible to bioinformatics experts and non-bioinformaticians alike
- 28. Sample to Insight QIAGEN Microbial Genomics Pro Suite (2) 28 Powered by quality components • Microbial Genomics Pro Suite expands upon the CLC Genomics Workbench • Plug-ins and modules add a layer of specialization: ◦ CLC Microbial Genomics Module ◦ CLC Genome Finishing Module ◦ MetaGeneMark plug-in • Upload data and using customizable workflows, data is just a few clicks away • Easy-to-use graphic user interface • Quick and helpful customer service
- 29. Sample to Insight • 16S: Greengenes/SILVA • WGS: GO Pfam Add database QIAGEN Microbial Genomics Pro Suite (3) 16S and WGS workflows Data import and QC Meta- genome assembly Meta- genome analysis OTU clustering Alpha, beta diversity • Trim and quality filter raw reads • Merge paired-end reads • De novo assembly • Identify gene and CDS • Annotate CDS • Build functional profile (GO) • Statistical analysis • Visualize data • Open and closed OTU picking • OTU table filtering • Tree building • Phylogenic diversity • UniFrac distances • Statistical analyses 29
- 30. Sample to Insight QIAGEN Microbial Genomics Pro Suite (4) 30 16S OTU and taxonomic assignment workflow • Includes quality filtering • For OTU picking, sequences were mapped against the greengenes database and clustered at 97% identity • Unmapped sequences were then clustered de novo • Results shown are summarized at the genus level • Highly customizable outputs
- 31. Sample to Insight QIAGEN Microbial Genomics Pro Suite (5) 31 Alpha and beta diversity workflow • Young individuals have a higher alpha diversity compared with old, as measured by observed OTUs • Other metrics include Shannon and Simpson Diversity, phylogenetic diversity • Highly customizable output • Beta diversity: Unweighted UniFrac analysis • Young and old gut communities are significantly different (P=0.047 Permanova analysis, corrected for multiple comparisons) • Other metrics include weighted UniFrac, generalized UniFrac, Bray-Curtis, Euclidean • Highly customizable outputs
- 32. Sample to Insight QIAGEN Microbial Genomics Pro Suite (6) 32 WGS read mapping and taxonomic assignment workflow • Includes quality filtering and read merging • The taxonomic profiler maps each read against the chosen database • Output is an abundance table including graphical representation • Highly customizable output • The graph shows a summary at the genus level • CLC Microbial Genomics Pro Suite can also display alpha and beta diversity analyses (not shown)
- 33. Sample to Insight QIAGEN Microbial Genomics Pro Suite (7) 33 WGS de novo assembly and functional assignment workflow • Includes quality filtering • De novo assembly of reads, identification and annotation of genes and coding sequences • Performs functional and statistical analyses • Highly customizable output • Samples are clustered by functional similarity using Euclidean distances • CLC Microbial Genomics Pro Suite can also perform taxonomic assignment (not shown)
- 34. Sample to Insight QIAGEN Microbial Genomics Pro Suite (8) 34 WGS de novo assembly and functional assignment workflow – differential abundance analysis Name Max group mean Log₂ fold change Fold change p value FDR p value 0008452 // RNA ligase activity 151.2 -1.64 -3.12 0.000216912 0.27 0071804 // cellular potassium ion transport 47 5.04 32.84 0.000765205 0.27 0071805 // potassium ion transmembrane transport 47 5.04 32.84 0.000799437 0.27 0016407 // acetyltransferase activity 1229.8 -0.38 -1.30 0.001037548 0.27 0016410 // N-acyltransferase activity 1039 -0.43 -1.35 0.001044564 0.27 0046353 // aminoglycoside 3-N-acetyltransferase activity 22.6 -7.56 -188.37 0.001054322 0.27 0034069 // aminoglycoside N-acetyltransferase activity 22.6 -7.56 -188.37 0.001060815 0.27 0008080 // N-acetyltransferase activity 1039 -0.43 -1.35 0.001068129 0.27 0008234 // cysteine-type peptidase activity 615.2 0.57 1.48 0.00138698 0.28 0006276 // plasmid maintenance 145.2 3.68 12.85 0.001412554 0.28 0010393 // galacturonan metabolic process 16.8 -7.10 -137.19 0.002238434 0.33 0016837 // carbon-oxygen lyase activity, acting on polysaccharides 16.8 -7.10 -137.19 0.002247484 0.33 0045490 // pectin catabolic process 16.8 -7.10 -137.19 0.002280259 0.33 0045488 // pectin metabolic process 16.8 -7.10 -137.19 0.002313312 0.33 0047487 // oligogalacturonide lyase activity 15.8 -7.02 -129.63 0.002642413 0.35 0006787 // porphyrin-containing compound catabolic process 15.2 -6.93 -121.66 0.003588363 0.38 0046149 // pigment catabolic process 15.2 -6.93 -121.66 0.003699807 0.38 0033015 // tetrapyrrole catabolic process 15.2 -6.93 -121.66 0.00381645 0.38 0015994 // chlorophyll metabolic process 15.2 -6.93 -121.66 0.003925426 0.38 0047746 // chlorophyllase activity 15.2 -6.93 -121.66 0.003935538 0.38 0015996 // chlorophyll catabolic process 15.2 -6.93 -121.66 0.003991415 0.38 0033926 // glycopeptide alpha-N-acetylgalactosaminidase activity 10.2 -7.30 -157.15 0.00434989 0.39 0036211 // protein modification process 1168.6 -0.43 -1.34 0.004493368 0.39 0006464 // cellular protein modification process 1168.6 -0.43 -1.34 0.004604395 0.39 0019439 // aromatic compound catabolic process 891.6 -0.51 -1.42 0.006839066 0.53 1901361 // organic cyclic compound catabolic process 890.6 -0.50 -1.42 0.006882415 0.53 0046700 // heterocycle catabolic process 890.6 -0.50 -1.42 0.007291632 0.54 0044270 // cellular nitrogen compound catabolic process 898.2 -0.50 -1.42 0.007732012 0.54 0009236 // cobalamin biosynthetic process 1097.4 -0.35 -1.28 0.007825894 0.54
- 35. Sample to Insight Semi-automated low-throughput workflow for microbial analyses 35 Summary • Semi-automated purification of high-quality, inhibitor-free DNA ◦ QIAamp PowerFecal kit (cat no. 12830-50) ◦ DNA purified, hands-free, on QIAcube in ~1 hour (cat. no. 9001882) ◦ DNA can be used immediately in downstream applications • Easy, fast, high-quality DNA library prep ◦ QIAseq 1-Step Amplicon Kit (16S) (cat. no. 180412/180415) ◦ QIAseq FX DNA Library Kit (WGS) (cat. no. 180473) • QIAGEN Microbial Genomics Pro Suite provides user-friendly, highly accurate microbial analysis ◦ 16S ◦ WGS
- 36. Sample to Insight Questions 36 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. Trademarks: QIAGEN®, Sample to Insight®, QIAamp®, QIAcube®, QIAseq™, DNeasy®, Inhibitor Removal Technology®, PowerFecal®, PowerSoil®, QuantiFast® (QIAGEN Group); Illumina®, HiSeq®, MiSeq® (Illumina, Inc). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. 05/2017 PROM-10910-002