Performance analysis of new sequencing technologies (ONT, PacBio) and genome assembly pipelines compared to Illumina

1. Genome Assembly from Three Sequencing Platforms:
MinION, MiSeq and PacBio
Francesca Giordano, Louise Aigrain, Michael Quail, James Bonfield,
Robert Davies, David Jackson, Thomas Keane, Zemin Ning and Richard Durbin

2. 2
Yeast strains: S288c, SK1, N44, CBS
S288c Reference:
12 Million bases,17 chromosomes
Sequenced at the Wellcome Trust Sanger Institute
MinION Reads
Strain
Bases
(Mb)
Reads
Mean
Length
Longest
Read
Coverag
e
Identity
Numbe
r of
Runs
Flowcell
S288c 323 32770 9843 56477 27X 93% 3 R7
N44 130 15654 8292 37837 11X N/A 4 R7
CBS 109 12211 8952 46481 9X N/A 4 R7
SK1 51 5938 8589 36791 4X N/A 2 R7
MiSeq reads, for each strain: ~120X coverage, Identity ~93%
ONT Read
Lengths
PacBio Read
Lengths
PacBio Reads
Strain
Bases
(Mb)
Reads
Mean
Length
Longest
Read
Coverag
e
Identity
Number
of Runs
S288c 1463 239408 6109 35196 120X 93% 3
N44 1794 371025 4834 33906 148X N/A 3
CBS 1639 324414 5052 34173 134X N/A 2
SK1 3019 697989 4325 34080 248X N/A 5

3. 3
Assemblers and other analysis tools
De Novo Assembly with Long Reads (ONT & PacBio)
Canu https://github.com/marbl/canu
Falcon https://github.com/PacificBiosciences/falcon
MiniAsm https://github.com/lh3/miniasm
PBcR http://wgs-assembler.sourceforge.net/wiki/index.php/PBcR
Nanopolish https://github.com/jts/nanopolish
Analysis I, S288c:
De novo Assembly with Long
reads only -- ONT or PacBio
Analysis II, all strains:
De novo Assembly with
MiSeq reads, Scaffolding using
Long Reads -- ONT or PacBio
More tools used:
Poretools (https://github.com/arq5x/poretools),
dnadiff (https://github.com/garviz/MUMmer)
De Novo Assembly with MiSeq Reads
SOAP denovo http://soap.genomics.org.cn/soapdenovo.html
Fermi https://github.com/lh3/fermi
SPAdes http://bioinf.spbau.ru/spades
Masurca http://www.genome.umd.edu/masurca.html
Scaffolding pipelines
Hybrid-SPAdes http://bioinf.spbau.ru/spades
SMIS https://sourceforge.net/projects/phusion2/files/smis/

4. 4
Read Length distribution
Analysis I: S288c, de novo assembly with Long Reads only
Nanopore vs. PacBio Platforms
S288c
Reads
Bases
(Mb)
Reads
Mean Read
Length
Longest
Read
Coverage Identity
Nanopore 323 32770 9843 56477 27X 93%
PacBio 328 34248 9584 32921 27X 92%

5. 5
Analysis I: S288c, de novo assembly with Long Reads only
Nanopore vs. PacBio Platforms
S288c
Reads
Bases
(Mb)
Reads
Mean Read
Length
Longest
Read
Coverage Identity
Nanopore 323 32770 9843 56477 27X 93%
PacBio 328 34248 9584 32921 27X 92%
Platform Assembler
Bases
(Mb)
Contigs
N50
(Kb)
Reference
coverage
Mismatches
(Kb)
Indels
(Kb)
Identity
CPU
Time (h)
Nanopore
Falcon 1.15 42 522 96.9% 7 279 97.5% 14
Canu 1.19 27 769 98.8% 2 229 98.0% 172
PBcR 1.35 101 528 99.2% 3 233 98.0% 496
MiniAsm 1.18 33 540 95.2% 431 820 89.2% 0.07
PacBio
Falcon 1.18 70 272 95.6% 4 295 99.7% 8
Canu 1.24 32 616 99.9% 1 10 99.9% 25
PBcR 1.25 37 751 99.2% 2 10 99.9% 52
MiniAsm 1.25 40 467 95.8% 232 1060 89.4% 0.05

6. 6
Additional tests performed
Analysis I: S288c, de novo assembly with Long Reads
only
De novo assembly with varied coverage: 10X, 20X, 27X
for both ONT and PacBio data
De novo assembly using 2D reads from Pass folder
versus using 2D reads from Pass+Fail folders
Polish assemblies with Nanopolish to improve accuracy
De novo assembly with the full PacBio data samples:
> 120X per strain
Analysis II: Scaffolding drafts assemblies from MiSeq
data
De novo assembly with MiSeq reads and compare results
of scaffolding by Hybrid-SPAdes and by the SMIS pipeline
De novo assembly using 2D reads from Pass folder
versus using 2D reads from Pass+Fail folders
Thank you!
Acknowledgments:
Louise Aigrain, Michael Quail, James Bonfield, Robert
Davies, David Jackson, Thomas Keane, Zemin Ning,
Richard Durbin and Gianni Liti

7. Backup slides

8. 8
Run details
Date Flowcell 2D ‘Pass’ 2D ‘Pass’ + 2D ’Fail’ 1D ‘Pass’ + 1D ’Fail’
Bases (Mb) Reads Bases (Mb) Reads Bases (Mb) Reads
S288c
18/02/2016 R7 53 4722 95 11631 252 32290
09/03/2016 R7 18 1937 30 3455 71 8535
11/03/2016 R7 111 11854 355 34586 676 87457
11/03/2016 R7 141 14257 252 27971 635 75977
N44
14/03/2015 R7 6 3101 14 4373 107 40519
20/04/2015 R7 1 154 1 233 12 1745
24/05/2015 R7 5 576 10 1166 39 6507
22/10/2015 R7 118 9026 186 22739 425 57893
CBS
24/05/2015 R7 6 700 9 1125 29 4497
24/05/2015 R7 15 1509 24 2725 80 11085
09/09/2015 R7 32 4066 42 5763 99 14531
22/10/2015 R7 56 5868 73 8159 169 21782
SK1
18/02/2016 R7 27 3166 143 19017 391 53137
10/03/2016 R7 24 2772 69 8990 176 24291
Run with the modified scripts by John Tyson (https://
wiki.nanoporetech.com/pages/viewpage.action?pageId=35523684)

9. 9
Analysis I: S288c, de novo assembly with Long Reads only
Nanopore Reads, 27X Coverage
Canu
PBcR MiniAsm
Falcon Canu
PBcR MiniAsm
Falcon
PacBio Reads, 27X Coverage

10. 10
Analysis I: S288c, de novo assembly with Long Reads only
Canu assembly from Nanopore polished
Assembler
Bases
(Mb)
Contigs
N50
(Kb)
Mismatches
(Kb)
Indels
(Kb)
Identity
CPU
Time (h)
Canu 1.19 27 769 1.8 229 98.0% 172
Canu+
Nanopolilsh
1.21 27 783 1.6 49.3 99.5% 873

11. 11
Analysis I: S288c, de novo assembly with Long Reads only: PacBio Reads
Assembler Bases (Mb) Contigs N50 Identity
S288c
120X
Falcon 11.88 24 804938 99.99%
Canu 12.33 21 783441 99.99%
PBcR 12.19 17 809591 99.99%
MiniAsm 12.47 29 668702 89.20%
N44 148X
Falcon 11.71 22 788656 N/A
Canu 11.94 20 800107 N/A
PBcR 11.91 22 799764 N/A
MiniAsm 12.06 17 829559 N/A
SK1 326X
Falcon 10.44 104 272457 N/A
Canu 12.32 22 798830 N/A
PBcR 12.45 33 829345 N/A
MiniAsm 12.34 22 839990 N/A
CBS 134X
Falcon 11.78 24 1475018 N/A
Canu 12.14 19 829217 N/A
PBcR 12.24 33 815875 N/A
MiniAsm 12.37 20 1525406 N/A

12. 12
Strain Pipeline Bases Scaffolds N50 Identity
Hybrid-SPAdes versus SPAdes + SMIS
S288c 27X
Spades +SMIS 11727537 97 494844 99.98%
Hybrid-Spades 11770749 66 444120 99.97%
N44 11X
Spades +SMIS 11695790 66 653753 N/A
Hybrid-Spades 11703568 58 348695 N/A
CBS 9X
Spades +SMIS 11695531 73 502933 N/A
Hybrid-Spades 11709420 45 548274 N/A
SK1 4X
Spades +SMIS 11671735 147 343333 N/A
Hybrid-Spades 11684067 112 226622 N/A
Best Assemblies: SOAP denovo + SMIS
S288c 27X SOAP k=81 11744509 83 818222 99.97%
N44 11X SOAP k=101 11663034 48 663565 N/A
CBS 9X SOAP k=101 11659507 69 793208 N/A
SK1 4X SOAP k=81 11623050 114 452917 N/A
Analysis II: De novo Assembly with MiSeq reads, Scaffolding using Long Reads
Nanopore Reads