ASM Microbe 2017: Reaching the Parts Other Methods Can't: Long Reads for Microbial Genomics and Metagenomics
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Long read sequencing technologies allow for complete microbial genome reconstruction from metagenomic samples. Single molecule long reads from PacBio and nanopore provide contigs but have high error rates. Synthetic long reads from 10X Genomics and other linked read methods generate more accurate scaffolds. While high DNA input requirements and extraction challenges remain, long reads are promising for de novo assembly, structural variation detection, and resolving complex populations without cultivation.
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ASM Microbe 2017: Reaching the Parts Other Methods Can't: Long Reads for Microbial Genomics and Metagenomics
- 1. Reaching the Parts Other Methods Can’t: Long Reads for Microbial Genomics and Metagenomics Prof Nick Loman Institute of Microbiology and Infection University of Birmingham
- 2. COI Disclaimer: Oxford Nanopore Technologies provided free of charge reagents in support of some of the studies presented here and paid an honorarium for NJL to speak at a company meeting.
- 3. Why bother with long reads? • De novo assembly – Generate finished references for resequencing projects – Detection of large scale structural variation – Plasmid, IS element, reconstruction – Metagenomics • Metabarcoding – Full length amplicons (16S, 18S, …) • Haplotyping – Amplicons – Strain reconstruction in complex mixtures
- 4. Long read technologies • Single molecule • Synthetic/linked
- 5. Long read technologies • Single molecule – Contigs – Noisy (5-15% error) – Best for assembly • Synthetic/linked – Scaffolds – Very accurate – Best for haplotyping
- 6. 10X Genomics Chromium: 1m GemCodesGemCode: 100k GemCodes James Hadfield, CRUK Very low input requirement (1ng) Long fragments needed >50kb 5 minute processing time
- 7. Patchy coverage within single molecule Bryan Wee, James Hadfield
- 8. Long reads for reference genoems
- 9. How long is long enough? • 7kb reads will bridge rRNA operon • Typically the longest bacterial repeat sequence Serge Koren, Adam Phillippy
- 10. Quick et al. BMJ Open 2014 http://bmjopen.bmj.com/content/4/11/e006278.long Forensic source tracking of Pseudomonas aeruginosa in a Burns unit
- 11. • ST395 representative sequence by PacBio (P4-C2) • Resolve to two contigs With thanks to Lex Nederbragt SNPs Indels Mapped PAO1 Reference 23 4 77% PacBio Reference 40 5 97% Choice of reference informs phylogenetic resolution
- 12. Bed 11 Environment Water Patient ΔoprD ΔlysR-like regulator Rapid emergence of meropenem resistance
- 13. Long reads for metagenomics
- 14. Noisy reads for metagenomics • PacBio – Even HMP mock community Courtesy of Pacific Biosciences
- 15. Long read technologies • Oxford Nanopore MinION • Portable, fieldable • Real-time • Low/no instrument cost • Reads up to 1Mb • 1-10Gb per run • Homopolymeric tract errors • 1D: 15% error • 1D^2: 5% error
- 16. Nanopore WGS Consortium, https://github.com/nanopore-wgs-consortium/NA12878 R9.4 chemistry enables higher yields
- 17. In field whole-genome sequencing MRSA S. pyogenes 900Mb 1D rapid P. aeruginosa E. faecium Bandage by Ryan Wick 4.4Gb 1D ligation
- 19. Whale watching with nanopore • Sambrook • Excess of DNA (>10ug) • V. careful pipetting • Aim <1 transposase cut per molecule Picture from Phil Zuzerte
- 20. Total bases: 5,014,576,373 (5Gb) Number of reads: 150,604 N50: 63,747 Mean: 33,296.44 http://lab.loman.net/2017/03/09/ultrareads-for-nanopore/ Whale watching: E. coli
- 21. 1113805 916705 790987 778219 771232 671130 646480 629747 614903 603565 Whale watching: E. coli
- 22. “Squiggles are vanity, alignments are sanity!
- 24. E. coli: genome assembly in 8 reads Read Length Ref start Ref end Time (m) 1 876991 4398844 634183 32.48 2 696402 470003 1166405 25.79 3 799047 1137438 1936485 29.59 4 642071 1759431 2401502 23.78 5 826662 2106227 2932889 30.61 6 883962 2699626 3583588 32.73 7 825191 3285196 4110387 30.56 8 463341 3995967 4459308 17.16 miniasm N50 4Mb Time: 1.5s (1 CPU) 1x coverage!
- 25. PFGE: The future
- 26. What’s coming next? • Combined single molecule and linked read hybrid metagenomic assemblies? • New methods for long fragment extraction from microbiome studies • Hi-C to link chromosomes w/ plasmids, phage • Much higher outputs
- 27. Conclusions • Long reads and synthetic long reads promising for whole-genome reconstruction from metagenomics • Synthetic long reads (10X and/or hybrid) will be useful for detecting low abundance members of population • Issues: – Very high input requirements may be challenging for certain applications, e.g. diagnostics from clinical samples – Extraction of high molecular weight DNA from mixed samples tricky; no one size fits all solution – Cost remains prohibitive for very deep sequencing
- 28. Acknowledgements – Josh Quick (Birmingham) – 10X: • Pablo Fuentes-Utrilla (Birmingham) • Bryan Wee, Ross Fitzgerald (Roslin) • James Hadfield (CRUK) – Nanopore human genome sequencing consortium – Pseudomonas • Nicola Cumley, Beryl Oppenheim
- 29. Synthetic long reads • Moleculo – Long range amplification – “Nextera on steroids” – 500ng input – Size selection required http://www.illumina.com/products/truseq-synthetic-long-read-kit.ilmn
Editor's Notes
- Water is an especially important aspect to burns care as part of the treatment is to have dressing changes and skin removal under the shower. The burns unit at the QE has several shock rooms in which are walk in shower areas. The invidual patients rooms have a walk in shower area with either a shower bed or trolly where the patient can be transferered to to have dressing changes and wound cleaned. Thease rooms are also kept very warm, up to 37o perfect incubation temperature for bugs! Pseudomonas oubreaks have previously been linked to hydrothreapy baths in the early 90’s.