Next-Generation Sequencing of Microbial Genomes and Metagenomes

1. Next-Generation Sequencing of Microbial Genomes and Metagenomes Christine KingFarncombeMetagenomics Facility Human Microbiome Journal Club July 13, 2012

2. Overview • Next-generation sequencing • Applications • Instruments • Library prep and sequencing chemistry • Sequence quality • Project overview • Microbial genomes • Microbial communities

3. DNA Sequencing • 1st generation • Sanger chain termination • Capillary electrophoresis • 2nd generation (NGS) • High throughput, “massively parallel” • Shorter reads • Sequencing-by-synthesis • 3rd generation • Single molecule • Nanopores

4. Applications • DNA sequencing • De novo genomes • Resequencing • Shotgun (e.g. mutant strains) • Amplicon (e.g. HLA, cancer) • Sequence capture (e.g. exome) • Metagenome • Amplicon (e.g. 16S, COI, viral) • Shotgun • ChIP • RNA sequencing • Gene expression • Gene annotation, splice variants • Metatranscriptome

5. Instruments

6. Instruments

7. Which instrument(s) to use? • Read length vs number of reads • Cost per base, per sample, per project (multiplexing?) • Accuracy • Run time, wait time

8. Library Preparation • Goal: fragments of DNA, each end flanked by adaptor sequences • Adaptors contain amplification- and sequencing primer binding sites; platform- and chemistry-specific • Optional: sample-specific barcodes/indexes/MIDs/tags allow multiplexing during sequencing • Library QC: quantity, size

9. Library Preparation • Library types: • Shotgun (DNA) • May begin with ChIP • May follow with sequence capture • Mate pair (DNA) • Amplicon (DNA) • Total RNA • May enrich for mRNA (poly-A enrichment, rRNA depletion) • Convert to cDNA (then similar to DNA protocols) • Small RNA • RNA ligations, convert to cDNA after

10. Library Preparation: Shotgun • Fragmentation • Sonication • Nebulization • Enzymatic • End repair • 3’ overhangs digested • 5’ overhangs filled • 5’ phosphate added

11. Library Preparation: Shotgun • Adapter ligation • T-overhangs • Forked structure controls orientation • Library amplification • Few cycles • Enrich for correctly-adapted fragments • Required to complete adapter structure in some protocols • Size selection • Gel excision, AMPure beads • Limit insert size as needed, remove artifacts

12. Library Preparation: Amplicon • Amplify region of interest using PCR • Primers contain adapter sequences

13. Library Preparation: Mate Pair • Begin with large fragments (e.g. 3kb, 20kb) • Circularize and fragment again • Illumina: direct ligation • 454: Cre/Lox recombination • Enrich for fragments containing the junction • Proceed with shotgun library prep

14. Library Preparation: Mate Pair • Why? Paired sequences are a known distance apart; improves genome assembly • Note: 454 calls these “paired end libraries”, not to be confused with Illumina’s “paired end sequencing”!

15. Sequencing: Illumina • Cluster generation • Library fragments hybridize to oligos on the flow cell • New strand synthesized, original denatured, removed • Free end binds to adjacent oligos (bridge formation) • Complimentary strand synthesized, denatured (both tethered to flow cell) • Repeat to form clonal cluster • Cleave one oligo, denature to leave ssDNA clusters • ~800K clusters/mm^2

16. Sequencing: Illumina • Variety of workflows: • Single- or paired end reads • 0, 1, or 2 index reads

17. Sequencing: Illumina • At each cycle, all 4 fluorescently-labeled nucleotides pass over the flow cell • Each cluster incorporates one nt (terminator) per cycle • Fluor is imaged, then cleaved • De-block and repeat

18. Sequencing: Illumina • Other terminology: • cBot – accessory instrument that performs cluster generation • Lanes – divisions (8) of HiSeq and GAIIx flow cells • PhiX – bacteriophage with small, balanced genome; PhiX library spiked in with samples for QC • Phasing/pre-phasing – nt incorporation falls behind or jumps ahead on a portion of strands in the cluster and contributes to noise • Chastity filter – measures signal purity (after intensity corrections); if the background signal is high, cluster will be discarded • BaseSpace – cloud computing site for processing MiSeq data • File format: fastq

19. Sequencing: 454 • emPCR: clonal amplification of bead-bound library in microdroplets • Library input amounts critical! • One molecule per bead • Titration procedure

20. Sequencing: 454 • Library capture: beads coated with complimentary oligo • Amplification: droplet contains PCR reagents and the other oligo • Post-PCR: millions of identical fragments attached to the bead

21. Sequencing: 454 • Bead Recovery: physical and chemical disruption • Enrichment: capture successfully amplified beads using biotinylated primers + magnetic, streptavidin beads

22. Sequencing: 454 • Deposit bead layers onto PicoTiterPlate: • Enzyme beads • Enriched DNA beads • More enzyme beads • PPiase beads

23. Sequencing: 454

24. Sequencing: 454 • Pyrosequencing • 4 nucleotides flow separately • If nt incorporation…PPi...light • APS + PPi (sulfurylase)  ATP • Luciferin + ATP (luciferase) light + oxyluciferin • Amount of light proportional to #nt incorporated • Rinse and repeat with next nt

25. Sequencing: 454 • Camera captures light emitted from every well during every nucleotide flow

26. Sequencing: 454 • Flowgram: representation of a sequence, based on the pattern of light emitted from a single well

27. Sequencing: 454 • Other terminology: • Lib-L/Lib-A: adapter variants, “ligated” or “annealed” • Titanium chemistry: ~450 bp reads on all instruments • XL+ chemistry: ~700 bp reads on the FLX+ instrument • Flow: one of the four nucleotides flows over the PTP • Cycle: a set of four flows, in order • Valley flow: if number of bases incorporated in a given read during that flow is uncertain, e.g. 1.5 units of light (background signal, homopolymers) • File format: sff (standard flowgram format)

28. Sequencing: Ion Torrent • Procedures and chemistry similar to 454 • Instead of PPi, measure H+ release (pH change) via semiconductor chip • No expensive camera or laser required, no modified nucleotides

29. Sequence Quality • Error probabilities determined using training sets, platform-specific biases • Expressed as a quality value (QV or Q score) per base • Similar to PHRED scores: • Q = -10 log10P • P = 10 -Q/10

30. Project 1: Microbial Genome • Considerations: • Reference genome? • How much coverage do I want? • How big is the genome • How much data do I need? • bp needed = genome size X coverage • Which instrument/chemistry configuration to use? • Coverage • Depth (number of times a particular base is “covered” by a read (e.g. 25X) • Breadth (% of genome with at least 1X coverage)

31. Project 1: Microbial Genome • Sample preparation • Isolate high quality (not degraded) and high purity (no RNA) gDNA • Verify on a gel • Quantify using dsDNA-specific dye • Library preparation • Can do this yourself if you like • ~ $200 per sample for Nextera • Cheaper protocols • Cheaper in bulk • Barcode compatibility

32. Project 1: Microbial Genome • Library QC • Insert size confirmed on BioAnalyzer (within range, no artifacts) • Poolbarcoded libraries (normalize based on PicoGreen quantification) • Absolute quantification of library pools using qPCR

33. Project 1: Microbial Genome • MiSeq sequencing • Dilute and denature library pool (optimal concentration requires titration...) • Spike in PhiX library as needed (e.g. 1%) • Prepare and load reagents, flow cell • Basic filtering and de-multiplexing performed automatically • Download fastq files from BaseSpace

34. Project 1: Microbial Genome • Data processing • Additional filtering • Trim the ends • Remove PCR duplicates • Assembly: overlapping reads are assembled to eachother based on sequence similarity = contigs

35. Project 1: Microbial Genome • What’s next? • Polish the genome (hybrid assemblies, mate pair libraries) • Annotate (ORFs, RNA-seq) • Compare

36. Project 2: Microbial Community • Shotgun metagenomics • Unbiased survey of community content • Random library fragments may provide very little taxonomic resolution (e.g. conserved, unknown) • Identify genes, classify by function • Targeted metagenomics • Limited survey of community content • Targeted loci provide excellent taxonomic resolution, but may exclude certain taxa • Identify OTUs, classify by taxonomy

37. Project 2: Microbial Community • 16S rRNA • Multi-copy gene (1.5 kb) • Conserved and hypervariable regions • Extensive databases from known species

38. Project 2: Microbial Community • Considerations: • Biases in sampling methods, culturing, DNA isolation, PCR...replicate • Available SOPs • How many reads per sample? • Read length matters! • Sample preparation: • Isolate DNA • PCR amplify, purify • High-fidelity polymerase • Barcoded primers • No primer dimers! • Normalize PCR products and pool

39. Project 2: Microbial Community • 454 Sequencing • emPCR titrations with different library input • Bulk emPCR • Sequence • Basic filtering • Collect sff files • Data processing • De-multiplexing • Additional filtering • Trim the barcodes, primers • Check for chimeras

40. Project 2: Microbial Community • Clustering • Sequences grouped by similarity = OTUs

41. Project 2: Microbial Community • Taxonomic identification • OTUs are classifed by comparing to known 16S sequences • Level of classification (e.g. family vs genus)? • Diversity • Within sample • Between samples