- 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. Plans are outlined to apply HiFi reads to structural variant benchmarking and expand small variant calling to difficult regions.

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.