1.
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved.
PacBio Circular Consensus Sequencing
of Genome in a Bottle samples
Aaron Wenger 2019-03-28

2.
PACBIO DATASETS FOR GIAB SAMPLES
Wang, Olson, et al. (2019). bioRxiv. doi:10.1101/562611
Sample Sequencer CLR Datasets
HG001 / NA12878 PacBio RS II 44-fold
Ashkenazim Trio PacBio RS II 70-fold for HG002, 30-fold for HG003 and HG004
Chinese Trio Sequel System 60-fold for HG005, 30-fold for HG006 and HG007
200 6040
Read length (kb)
Coverage(cumulative)
0
10
20
30
40
50
HG005
HG006
HG007

3.
PACBIO CONTINUOUS LONG READS (CLR)
NGS
PacBio CLR
Raw Reads IGV “Quick Consensus”

4.
PACBIO CIRCULAR CONSENSUS SEQUENCING (CCS)
Double-stranded DNA
Ligate adapters
Anneal primer and bind
DNA polymerase
Sequence
Generate
consensus HiFi read
Subreads
(passes)
Subread errors
Wenger, Peluso, et al. (2018). bioRxiv. doi:10.1101/519025

5.
PACBIO CIRCULAR CONSENSUS SEQUENCING (CCS)
Double-stranded DNA
Ligate adapters
Anneal primer and bind
DNA polymerase
Sequence
Generate
consensus HiFi read
Subreads
(passes)
Subread errors
Passes
5 10 15 200
30
0
10
20
40
50
Accuracy(Phred)
Wenger, Peluso, et al. (2018). bioRxiv. doi:10.1101/519025
Read accuracy improves
with more passes

6.
HIFI READS ARE LONG AND ACCURATE
Wenger, Peluso, et al. (2018). bioRxiv. doi:10.1101/519025
10 15 20
50 60
Read accuracy (Phred)
Read length (kb)
403020
5
0
100,000
200,000
300,000
0
100,000
200,000
ReadsReads
Avg.
13.5 kb
Avg.
99.8%
(Q27)

7.
HIFI READS ARE LONG AND HIGHLY ACCURATE

8.
HG002 HIFI DATASETS
Coverage Average Read Length
30-fold 9.6 kb
28-fold 13.5 kb
- Reads on SRA and NIST FTP
- Alignments on NIST FTP
Richard Hall, Paul Peluso, Yufeng Qian, David Rank, Billy Rowell
https://bit.ly/2THv47q (NIST FTP)

9.
Sequel System (1M)
Sequel II System (8M)
Subread Yield 318 Gb
CCS Yield 16 Gb
CCS Accuracy 99.8%
Read length (kb)
0 50 100 150 200 250 300
Yieldperunitreadlength(kb)
0
25
50
100
150
175
75
125
SEQUEL II SYSTEM

10.
Sequel System (1M)
Sequel II System (8M)
Subread Yield 318 Gb
CCS Yield 16 Gb
CCS Accuracy 99.8%
Read length (kb)
0 50 100 150 200 250 300
Yieldperunitreadlength(kb)
0
25
50
100
150
175
75
125
Accuracy(Phred)
5 10 15 200
30
0
10
20
40
50
Sequel (1M)
Passes
30
0
10
20
40
50
85 15 200 10
Passes
Sequel II (8M)
SEQUEL II SYSTEM

11.
PLANS FOR NEXT QUARTER
Sample Sequencer CCS Dataset
HG002 Sequel System 30-fold with 9.6 kb reads
HG002 Sequel System 28-fold with 13.5 kb reads
HG002 Sequel II System 30-fold with 11.0 kb reads
HG001 Sequel II System 30-fold with 10 kb reads
HG005 Sequel II System 30-fold with 10 kb reads
Submitted
In Progress
Primo Baybayan, Shreya Chakraborty, Alicia Yang

12.
APPLICATIONS OF HIFI READS FOR GENOME IN A BOTTLE
Structural variant benchmark
-HG002: HiFi structural variant callset rivals multi-technology benchmark
-Extend to HG001, HG005
Small variant benchmark (v4α for all samples)
-Expand to difficult regions
-Correct mistakes
Phasing variants

13.
VARIANT CALLING AND PHASING FOR STRC

14.
SUMMARY
bioRxiv 519025
doi:10.1101/519025
Baylor – Medhat Mahmoud, Fritz Sedlazeck
Dana-Farber – Heng Li
Chinese Academy of Agricultural Sciences – Jue Ruan
DNAnexus – Chen-Shan Chin, Arkarachai Fungtammasan
Google – Andrew Carroll, Pi-Chuan Chang, Mark DePristo,
Alexey Kolesnikov
Johns Hopkins – Michael Alonge, Michael Schatz
Max Planck Dresden – Gene Myers
NIH/NHGRI – Sergey Koren, Adam Phillippy
NIST – Nathan Olson, Justin Zook
PacBio – Greg Concepcion, Richard Hall, Paul Peluso, Yufeng Qian, David Rank, William
Rowell, Armin Töpfer, Aaron Wenger
Saarland University – Jana Ebler, Tobias Marschall
-PacBio HiFi reads are long (>10 kb) and accurate (>99%).
-HiFi reads are available now for HG002 and soon for HG001 and HG005.
-HiFi reads will be useful for comprehensive variant detection and phasing.