Long read sequencing - LSCC lab talk - fri 5 june 2015
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Long read sequencing technologies such as Pacific Biosciences and Oxford Nanopore offer several advantages over short read technologies. They can generate reads of over 100kb which allows for untangling of repeats and completion of genomes and phasing of haplotypes. While PacBio is more established, Nanopore offers the potential for real-time, portable sequencing. Both require adaptation of bioinformatics tools and analysis approaches. The new technologies will change genomics jobs by moving more to streaming analysis and requiring skills in adapting to changing technologies.
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Long read sequencing - LSCC lab talk - fri 5 june 2015
- 1. Long read sequencing Torsten Seemann VLSCI LSCC Lab Talk - Melbourne, AU - Fri 5 June 2015 The good, the bad, and the really cool.
- 2. Why do we need long reads?
- 3. Repeats!
- 4. Long reads untangle graphs
- 7. Structural variation The missing heritability - not just SNPs & indels
- 9. Pacific Biosciences RSII 2015 ARC LIEF w/ Tim Stinear Installed this week. Passed testing!
- 10. Oxford Nanopore MinION MkI Successor to Mk0 MinION Access Program Round 2 The up & comer!
- 11. PacBio It’s already here and it works.
- 12. PacBio - the device ∷ It’s big! ∷ Three chunks : compute (left) : robotics (top) : sequencing (bottom) ∷ A cushion of N2 gas
- 13. PacBio - technology ∷ Polymerase bound to bottom of ZMW μ-well ∷ Fluorescent nucleotide incorporation measured in real time ∷ 3 hour “movies”
- 14. PacBio: read lengths Needs careful library prep to ensure DNA is not overly fragmented!
- 15. PacBio: error rate Single read: 86% 30x Consensus: 99.999%
- 16. PacBio: main applications ∷ Finished microbial genomes ∷ Full length cDNA (mRNA isoforms) ∷ Extreme GC sequence ∷ HLA / MHC / KIR haplotyping ∷ Base modifications (methylation)
- 17. PacBio: bioinformatics ∷ All in GitHub ∷ SMRT Portal : Nice GUI : Cloud ready : Linux backend : Cluster ready ∷ Cmdline too!
- 18. Oxford Nanopore The new kid on the block.
- 19. MinION - the device
- 20. PromethION - large scale ∷ 48 separate flow cells ∷ On board ASIC ∷ Runs Python
- 22. Nanopore - types of reads “1D reads” ∷ Template 1D ﹕ only fwd stran ∷ Complement 1D ﹕ only rev strand “2D reads” ∷ Normal 2D ﹕ mostly fwd, some rev ∷ Full 2D ﹕ most of fwd & rev ﹕ these are high quality
- 23. Nanopore - read lengths Read length is not limited by technology but by library preparation. Can get >100kbp reads. Read length
- 24. Nanopore - error rate ∷ 5-mer errors ∷ Not modelling base mods yet ∷ Basically where PacBio was a few years ago! Percent identity (aligned)
- 25. MinION - applications ∷ Same as PacBio plus.... ∷ Portable sequencing : in the field eg. Josh Quick in Guinea for Ebola : in hospitals - infection control : monitoring - water/food supply, production facilities : at the GP - pathogen test in 10 min from blood prick? : spit in a home device every morning?
- 26. MinION - bioinformatics ∷ Event space -vs- base space : MinION MkI - base calling in cloud (Metrichor) : MinION MkII - on device? : PromethION - can choose on-device add-on ∷ Mostly 3rd-party tools - lots of activity : poretools, poRe : minoTour, nanoPolish
- 27. Disruptive technology Just another sequencer?
- 28. “Run until” Dynamically adjust sequencing yield
- 29. “Read until” ∷ Can access events/bases during reading : remember reads are long 40 kbp : examine first 100 bp say : can decide to stop reading and eject molecule! ∷ This is a killer app! : only want pathogens? eject if human DNA : only want exome? eject if not exonic looking : controlled with Python code
- 30. VolTRAX - library prep
- 31. A new business model ∷ No capital or reagent costs : Instrument will be free : Flow cells will be free : Only pay for what you want to sequence : Min. $20 and ~$1000 for a 100x human genome ∷ But I’ll scam the system! : Flowcell stats sent back to base : Won’t send you new flow cells if they look unused
- 32. How will our job change?
- 33. Some things never change ∷ Don’t worry! : 50% of our job will always be converting file formats ☺ ∷ But things are improving : Pacbio: HDF5 : MinION: HDF5 / FAST5 ∷ Can convert .h5/.hd5 to .fastq easily
- 34. Read alignment ∷ PacBio : BLASR - Basic Local Alignment + Successive Refinement : BWA MEM - bwa mem -x pacbio ∷ MinION : MarginAlign - sum over possible alignments, HMMs : BWA MEM - bwa mem -x ont ∷ Need to modify variant caller parameters
- 35. De novo assembly ∷ Pacbio : HGAP, HGAP2, Falcon, Spades, Celera Assembler ∷ MinION : Spades, Celera Assembler, NanoPolish ∷ Lots of convergence : Similar error models (indels) : Long reads, lower coverage - back to the future!
- 36. Streaming analysis ∷ We are not going to keep all this data ∷ Extract info we need and discard ∷ Cheaper to resequence? ∷ Need to think streaming analyses ∷ Lots of new applications
- 37. Conclusion
- 38. Exciting times! ∷ Genomics is changing all the time : new technologies : changing attributes/properties of current technology ∷ Bioinformaticians need to be able to adapt : focus on key skills not specific apps ∷ Pipelines are often short lived : except maybe clinical / accredited ones
- 39. Contact ∷ tseemann.github.io ∷ t.seemann@unimelb.edu.au ∷ @torstenseemann