Error Correction and Assembly of Oxford Nanopore Sequencing

James Gurtowski

Assembly Complexity

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Assembly Complexity

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The advantages of SMRT sequencing Roberts, RJ, Carneiro, MO, Schatz, MC (2013) Genome Biology. 14:405

Oxford Nanopore MinION •  Thumb drive sized sequencer

powered over USB

•  Senses DNA by measuring changes to ion flow

•  Reads both DNA Strands (2D)

Nanopore Basecalling

Basecalling currently performed at Amazon with frequent updates to algorithm

Our Data - Yeast W303 Best: 446Mb

Mean Read Length: ~6kb (10kb shear)

New Flowcells 7k Avg Read Length

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Oxford Flowcell Yields

Flowcell YieldRead Length

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Nanopore Alignments

Alignment Statistics (BLASTN) Mean alignment length at ~7kbp

Shearing targeted 10kbp 255k reads align (64%)

174x coverage

7kb mean alignment length

68% mean identity

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Percent Identity

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Nanopore Accuracy Alignment Quality (BLASTN) Of reads that align, average ~65% identity “2D base-calling” improves to ~77% identity

65% AVG ID

77% AVG ID

ONT Read Alignments

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Partial Alignments

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Nanopore Alignment Summary 64% of the data map using BLASTN

Long Read Correction Algorithms PacBioToCA & ECTools

Hybrid Error Correction

Koren, Schatz, et al (2012) Nature Biotechnology. 30:693–700

HGAP & Quiver

LR-only Correction & Polishing

Chin et al (2013) Nature Methods. 10:563–569

PBJelly

Gap Filling and Assembly Upgrade

English et al (2012) PLOS One. 7(11): e47768

< 5x > 50x Long Read Coverage

NanoCorr: Nanopore-Illumina Hybrid Error Correction

1.  BLAST Miseq reads to all raw Oxford Nanopore reads

2.  Select non-repetitive alignments

○  First pass scans to remove “contained” alignments

○  Second pass uses Dynamic Programming (LIS) to select set of high-identity alignments with minimal overlaps

3.  Compute consensus of each Oxford

Nanopore read ○  Currently using Pacbio’s pbdagcon

https://github.com/jgurtowski/nanocorr

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Mean: 97%

Mean: 68%

Post Correction Identity

ONT vs Illumina Assembly

Oxford N50 : 585kb

Illumina N50 : 58kb

ONT Assembly Completeness

ONT Assembly Completeness

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LTR retrotransposon (5836 bp)

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Genomic Features (Average Length in bp)

S288CNanopore

Miseq

Long Read Assembly S288C Reference sequence •  12.1Mbp; 16 chromo + mitochondria; N50: 924kbp

Oxford Nanopore 28x corrected reads > 7kb NanoCorr + Celera Assembler •  95 non-redundant contigs •  N50: 585kbp >99.78% id

Illumina MiSeq 30x, 300bp PE (Flashed) Celera Assembler •  6953 non-redundant contigs •  N50: 59kb >99.9% id

Pacific Biosciences 25x corrected reads > 10kb HGAP + Celera Assembler •  21 non-redundant contigs •  N50: 811kb >99.8% id

E. Coli K12 Single Contig Assembly with MinION

Sequencing Data From: A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer Joshua Quick, Aaron R Quinlan and Nicholas J Loman

Single Contig Assembly 99.99% Identity (Pilon polishing)

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E. coli Error Correction with Nanocorr

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Nanocor Correction Results 145x Oxford Nanopore X 35x MiSeq

Future of Oxford Nanopore

Acknowledgements

Michael Schatz

Dick McCombie

Sara Goodwin

Schatz Lab

Oxford Nanopore Sequencing and de novo Assembly of a Eukaryotic Genome Sara Goodwin , James Gurtowski , Scott Ethe-Sayers , Panchajanya Deshpande , Michael Schatz , W Richard McCombie doi: http://dx.doi.org/10.1101/013490