Microbiome Isolation and DNA Enrichment Protocol: Pathogen Detection Webinar Series Part 2
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This slidedeck presents an easy-to-use workflow that allows selective isolation of microbial DNA from samples that are intrinsically rich in host DNA. This protocol includes steps for efficient depletion of host DNA while providing optimized conditions specific for bacterial lysis. This workflow is also specific for the identification of live bacteria, avoiding false results due to nucleic acids from dead bacteria. Enriched microbial DNA can be directly used in other molecular methods such as whole genome sequencing, qPCR and microarray assays.
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Microbiome Isolation and DNA Enrichment Protocol: Pathogen Detection Webinar Series Part 2
- 1. Sample to Insight Microbiome: Isolate and enrich microbial DNA with our new protocol isolate and enrich microbial DNA 1 Marco Polidori, Ph.D. Global Product Manager
- 2. Sample to Insight Welcome! 2 Pathogen detection: Microbial DNA isolation and identification to characterization • Part 1: Host-pathogen interactions: Molecular basis and host defense mechanisms • Part 2: Microbiome: Isolate and enrich microbial DNAwith our new protocol • Part 3: Microbiome: From identification to characterization isolate and enrich microbial DNA
- 3. Sample to Insight Legal disclaimer 3 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. isolate and enrich microbial DNA
- 4. Sample to Insight Agenda 4isolate and enrich microbial DNA Introduction • Host-microbe interactions • Microbiome definition and background • Technologies to analyze microbial communities Data: QIAamp® DNAMicrobiome protocol • Depletion of host DNA • 16S rRNA gene sequencing data of the human oral microbiome • Maintenance of community composition Summary • Improved microbiome sequencing through host DNA removal Questions 1 2 3 4
- 5. Sample to Insight Agenda 5isolate and enrich microbial DNA Introduction • Host-microbe interactions • Microbiome definition and background • Technologies to analyze microbial communities Data: QIAamp® DNAMicrobiome protocol • Depletion of host DNA • 16S rRNA gene sequencing data of the human oral microbiome • Maintenance of community composition Summary • Improved microbiome sequencing through host DNA removal Questions 1 2 3 4
- 6. Sample to Insight Microbiome: Definition and background 6isolate and enrich microbial DNA “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) What does “microbiome” mean? Kuczynski et al. Nature Reviews Genetics 13,47-58 (January 2012)
- 7. Sample to Insight Microbiome: Host-pathogen interaction 7isolate and enrich microbial DNA Microbiota and the host peacefully coexist to achieve a mutually beneficial relationship When the mutualistic relationship is disrupted, the microbiota can cause or contribute to diseases Nature.com Hybiske & Stephens Nature Reviews Microbiology 6, 99-110 (February 2008) Host-pathogen interaction Interaction of microbial communities and impact on human and animal health
- 8. Sample to Insight Microbiome: Definition and background 8isolate and enrich microbial DNA Consider this: • We have about 10 times as many microbial cells as human cells • 20% to 60% of all species may be uncultivable (The NIH HMP Working Group) • Each area of the body has its own microbiome (gut, mouth, skin etc.) • Microbiomes can be linked to many diseases (cancer, infections, metabolic disease) Hallmark microbiome features
- 9. Sample to Insight Microbiome: Definition and background 9isolate and enrich microbial DNA • 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 Disease • Blood • Sepsis/bloodstreaminfections • Oral • Periodontitis • Gingivitis • Neuronal • Anxiety • Depression Association of the human microbiota and disease http://commonfund.nih.gov/hmp/index
- 10. Sample to Insight Technologies to analyze microbial communities 10isolate and enrich microbial DNA • Gene cloning (Pan 16S rRNA) and sanger sequencing • Microarray • MALDI • Next generation sequencing • 16S rRNA sequencing • Whole genome sequencing • qPCR - Target dependent • 16S rRNA gene • Other relevant gene (antibiotic resistance gene, virulence factor gene)
- 11. Sample to Insight Technologies to analyze microbial communities 11isolate and enrich microbial DNA Next generation sequencing 16S rDNA sequencing • What species are present? • At what ratio? • How do they react to changes? • Correlation with health/disease status? Whole metagenome shotgun sequencing • Detailed analysis of unculturable microbes • Novel species/strains • Identify virulence factors (disease-related genes) • Functional analysis of bacterial community/metabolic networks qPCR - Target dependent • 16S rRNA gene • Other relevant gene (antibiotic resistance gene, virulence factor gene)
- 12. Sample to Insight Technologies to analyze microbial communities 12isolate and enrich microbial DNA • Methods for microbiome analyses: • 16s rRNA gene sequencing for community composition • Workflow: Mixed sample DNA isolation — host and microbes 16S rDNA amplification Amplicon sequencing Community composition Diversity within and in between samples Ashelford et al., Appl. Environ. Microbiol.71 7724-7736 (December 2005)
- 13. Sample to Insight Technologies to analyze microbial communities 13 • Methods for microbiome analyses: • Whole metagenome shotgun sequencing for additional information regarding function • Workflow: Comparison to reference databases and/or genomes Mixed sample DNA isolation — host and microbes Library preparation from fragmented DNA Whole genome sequencing Community composition Genome assemblyPresence of metabolic pathways and functions De novo assembly isolate and enrich microbial DNA
- 14. Sample to Insight Technologies to analyze microbial communities 14 Learnings from the HMP • Human Microbiome Project: • Up to 99 % human reads depending on sample material • Cost intensive waste of sequencing capacity • Additional filtering to obtain microbial data sets • Lower sequencing depth for microbial DNA of interest Human QC filtered Usable Percentage% A framework for human microbiome research, Nature. 2012 June 13; 486(7402): 215–221.(7402): 215–221. isolate and enrich microbial DNA
- 15. Sample to Insight Technologies to analyze microbial communities 15 DNA extraction can introduce community composition bias Willner et al. PLOS One, 2012; DOI: 10.1371/journal.pone.0034605 A bias in community composition may be introduced by sample preparation depending on: • Cell wall morphology (e.g., Gram, Actinomycetes, EPS) • Lysis methods used (e.g., mechanical vs. enzymatic) • Sample type (solid, viscous, host cell rich) isolate and enrich microbial DNA
- 16. Sample to Insight Agenda 16isolate and enrich microbial DNA Introduction • Host-microbe interactions • Microbiome definition and background • Technologies to analyze microbial communities Data: QIAamp® DNAMicrobiome protocol • Depletion of host DNA • 16S rRNA gene sequencing data of the human oral microbiome • Maintenance of community composition Summary • Improved microbiome sequencing through host DNA removal Questions 1 2 3 4
- 17. Sample to Insight Human DNA depletion: QIAamp DNA Microbiome 17 Overview sample preparation for whole metagenome shotgun sequencing: • Mechanical dispruption • Chemical lysis • Mild lysis conditions to keep bacteria intact • QIAamp protocol • All in one protocol for sample preparation and depletion of host DNA in about 180 minutes • Sample material: • Inherently rich in host DNA: buccal swabs, saliva, BAL, tissue, sputum, vaginal and nasopharyngeal swabs etc. • Downstream application: • Whole metagenome shotgun sequencing • 16S rRNA gene sequencing • qPCR isolate and enrich microbial DNA Host cell lysis Degradation of host cell nucleic acids Microbial cell lysis Isolation of microbial DNA Library preparation
- 18. Sample to Insight Human DNA depletion: Data 18 Quantification of human DNAdepletion • Reduction of human reads from over 90% to under 5% • At least 20x enrichment of sequences derived from the oral flora 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Lib 1 AHL Lib 2 QIAamp Lib 3 Molzym Percentreads Successful depletion of host DNA with the QIAamp DNA Microbiome protocol Human Oral Microbiome human QIAamp DNA Microbiome No host DNA depletion Kit 1 Host DNA depletion Kit 2 • Pooled buccal swab samples • GeneRead Library Preparation • 2x151 bp Illumina® MiSeq ® • FastQC for quality control • Mapping to hg19 with Bowtie 2 isolate and enrich microbial DNA
- 19. Sample to Insight Human DNA depletion: Data 19 Retainment of community composition Species Gram %GC Size [Mb] CFU persample STDEV Bacillus subtilis + 43.5 4.22 1.08E+05 2.16E+03 Corynebacterium glutamicum + 53.8 3.31 1.32E+05 5,72E+03 Escherichia coli - 50.8 4.56 1.24E+05 5,79E+03 Micrococcus luteus + 73.0 2.5 7.11E+04 9,05E+03 Rhodococcus erythropolis + 62.3-62.5 6.4-6.9 1.86E+05 1,65E+04 Xanthomonas campestris - 65.1 5.08 9.64E+04 4,44E+03 Whole Metagenome Shotgun Sequencing: • Quantify depletion of host DNA • Determine maintenance of bacterial community composition Sample Preparation: • Samples: buccal swabs with 7.2 x 105 colony forming units of mock community per sample • Comparison to alternative protocols isolate and enrich microbial DNA Buccal swab swirled in PBS Addition of mixed community Isolation of genomic DNA QIAGEN GeneRead Library Prep 2x151 bp Illumina MiSeq Count CFUs for each species
- 20. Sample to Insight 20 Sequencing results QIAamp Microbiome Kit 1 Kit 2 Kit 3 B. subtilis 4.66E+04 1.38E+04 3.61E+04 1.15E+04 C. glutamicum 2.28E+05 8.01E+03 2.34E+03 3.94E+03 E. coli 2.05E+05 1.50E+04 8.10E+03 1.38E+04 M.luteus 6.02E+04 3.45E+04 1.37E+05 3.88E+04 R.erythropolis 3.76E+05 1.61E+04 3,63E+03 8.66E+03 X.campestris 1.14E+05 1.10E+04 8,56E+03 1.22E+04 unmapped 8.34E+06 4.98E+05 6,12E+05 2.97E+05 Human hg19 4.18E+05 1.07E+07 6,44E+06 6.77E+06 total reads 9.79E+06 1.13E+07 7.24E+06 7.15E+06 total reads on bacterial references 1.03E+06 9.84E+04 1.95E+05 8.89E+04 Data Analysis (about 35.5 Mio reads): • Quality control (FastQC) • Mapping against reference genomes (Bowtie 2) • Normalization to genome size for community composition data • Ongoing Blast search for reads not mapping to reference genomes (oral microbiome) Human DNA depletion: Data isolate and enrich microbial DNA
- 21. Sample to Insight Human DNA depletion: Data 21 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Lib4-QIAamp DNA Microbiome Lib8-MoBio percentreads Host DNA depletion allows deeper analysis of microbial community X. campestris R.erythropolis M. luteus E. coli C. glutamicum B. subilits Human Oral Microbiome Human QIAamp DNA Microbiome No host DNA depletion Kit 3 Quantification of human DNAdepletion Depletion of host DNA provides valuable data for in depth analysis of the oral microbiome. Further analyzed by 16S isolate and enrich microbial DNA
- 22. Sample to Insight 16S rRNA gene sequencing — data 22 16s rRNA gene sequencing Caporaso et al., The ISME Journal (2012) 6, 1621–1624 • Buccal swab samples • 10 ng DNA in 16S rDNA PCR • Modified 515f PCR Primer and 806r PCR primer • 2x151 bp on Illumina MiSeq • Mapping with Bowtie2 to reference data from RDP database Fwd Primer 5‘ Rev Primer Adapter Adapter V4 BC Read 1 Index Read Read 2 3‘ BC 5‘3‘ About 350 bp isolate and enrich microbial DNA
- 23. Sample to Insight 16S rRNA gene sequencing: Data 23 16s rRNA gene sequencing Caporaso et al., The ISME Journal (2012) 6, 1621–1624 Depletion of human DNA improves amplification/yield of V4 16S region • PCR amplicons with modified 515f and 806r PCR primer • 25 cycles -750 -500 -250 -1000 bp QIAamp DNA Microbiome QIAamp DNA Microbiome Kit 3 Kit 1 Kit 2 Sample 2Sample 1 isolate and enrich microbial DNA
- 24. Sample to Insight 16S rRNA gene sequencing: Data 24 0% 20% 40% 60% 80% 100% Sample 1 Sample 2 Detection of bacterial families corresponding to the human oral microbiome Cardiobacteriaceae Coriobacteriaceae Bacteroidaceae Clostridiales Family XIII. Incertae Sedis Bacillaceae Erysipelotrichaceae Peptostreptococcaceae Flavobacteriaceae Campylobacteraceae Eubacteriaceae Lachnospiraceae Enterococcaceae Prevotellaceae Fusobacteriaceae Porphyromonadaceae Leptotrichiaceae Actinomycetaceae Carnobacteriaceae Micrococcaceae Neisseriaceae Streptococcaceae Veillonellaceae Pasteurellaceae Corynebacteriaceae DNA from buccal swabs isolated with the QIAamp DNA Microbiome protocol V4 Sequencing confirms representatives of the oral microbiome isolate and enrich microbial DNA
- 25. Sample to Insight Maintenance of community composition — data 25 Comparisonmapped reads to colony forming units in input material 0% 20% 40% 60% 80% 100% 120% Cfus in input sample QIAamp Microbiome Q M PS readsmappedtoreferencegenome Community composition with QIAamp DNAMicrobiome protocol X. campestris R. erythropolis M. luteus E. coli C. glutamicum B. subtilis Best correlation for community composition with QIAamp DNA Microbiome protocol No host DNA depletion Kit 3 Host DNA depletion Kit 2 No host DNA depletion Kit 1 QIAamp DNA Microbiome CFUs per sample Improved microbiome sequencing through host DNA removalisolate and enrich microbial DNA
- 26. Sample to Insight Summary: QIAamp DNA Microbiome main points 26 Improved microbiome sequencing through hostDNAremoval • Perfect for samples that contain human or animal cells (like swabs, body fluids) • Depletes >95% host DNA • Enables whole genome sequencing • High sensitivity in 16S analysis • Thorough lysis protocols minimize bias • Contains QIAamp UCP columns isolate and enrich microbial DNA
- 27. Sample to Insight QIAamp DNA Microbiome Kit specifics 27 Features Catalogue number: 51704 Time to extract: ca. 3h total Number of reactions: 50 Material included: QIAamp UCP columns, Pathogen Lysis Tubes, buffers, enzymes, 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.comor can be requested from QIAGEN TechnicalServices or your local distributor. Trademarks: QIAamp® , GeneRead™ (QIAGEN Group); Illumina® , MiSeq® (Illumina, Inc.). isolate and enrich microbial DNA
- 28. Sample to Insight Thank you for coming isolate and enrich microbial DNA Contact QIAGEN Technical Service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 for EU Email: techservice-na@qiagen.com techservice-eu@qiagen.com Qiawebinars@qiagen.com