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- 1. Next-Generation Sequencing of Microbial Genomes and Metagenomes Christine King Farncombe Metagenomics 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
- 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
- 5. Instruments
- 6. Instruments Total # of Read Cost outp Run Instrument read length per Technology ut Time s (bp) base (Gb) GS FLX 1M 450 0.5 $$$$ ++ GS FLX+ 1M 650 0.6 $$$$ ++ emPCR, SBS, light detection GS Jr 100K 450 0.05 $$$$ ++ GAIIx 640M 2x 150 90 $$ +++ HiSeq 2000 6B 2x 100 600 $ +++ Bridge PCR, SBS, fluororphore MiSeq 12M 2x 150 2 $$ ++ PacBio RS >10K >1000 0.01 $$$$ + Single-molecule seq, fluorophore SOLiD 5500xl 1.4B 75 + 35 155 $ +++ emPCR, probe ligation, fluorophore Ion PGM - 1M >100 0.1 $$$ + 316 emPCR, SBS, pH change Ion PGM - 6M >100 1 $$ + 318
- 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 Application Lengt # Accura Instruments Considerations h Reads cy De novo +++ ++ ++ MiSeq, 454, Ion Mix lengths (small) De novo +++ +++ ++ HiSeq, 454, Mix lengths, MP (large) SOLiD Re-seq ++ ++ ++ MiSeq, Ion Multiplex? (small) Re-seq (large) ++ +++ ++ HiSeq, SOLiD Enrichment? RNA-seq + +++ + Illumina, SOLiD, Ref? Size? (count) Ion Rare?
- 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 Primers contain interest using PCR 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: Enrichment: capture physical and successfully chemical disruption 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 Phred (Q) Probabilit Base Call Error probabilities Score y of Error Accuracy determined using (P) training sets, 10 1 in 10 90% platform-specific 20 1 in 100 99% 30 1 in 1K 99.9% biases 40 1 in 10K 99.99% Expressed as a 50 1 in 100K 99.999% 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: Coverage Reference genome? Depth (number of How much coverage times a particular do I want? base is “covered” by a read (e.g. 25X) How big is the genome Breadth (% of genome with at least 1X How much data do I coverage) need? bp needed = genome size X coverage Which instrument/chemistry configuration to use?
- 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 Insertsize confirmed on BioAnalyzer (within range, no artifacts) Pool barcoded libraries (normalize based on PicoGreen quantification) Absolute quantification of library pools using qPCR
- 33. Project 1: Microbial Genome MiSeq sequencing Diluteand 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 Assembly: Additional filtering overlapping reads Trim the ends are assembled to Remove PCR eachother based on duplicates 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 Targeted metagenomics metagenomics Unbiased survey of Limited survey of community content community content Random library Targeted loci provide fragments may excellent taxonomic provide very little resolution, but may taxonomic resolution exclude certain taxa (e.g. conserved, unknown) Identify OTUs, classify Identify genes, by taxonomy classify by function
- 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: Sample preparation: Biases in sampling Isolate DNA methods, culturing, PCR amplify, purify DNA isolation, High-fidelity PCR...replicate polymerase Available SOPs Barcoded primers How many reads per No primer dimers! sample? NormalizePCR Read length products and pool matters!
- 39. Project 2: Microbial Community 454 Sequencing Data processing emPCR titrations De-multiplexing with different library Additionalfiltering input Trim the barcodes, Bulk emPCR primers Sequence Check for chimeras Basic filtering Collect sff files
- 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