Slides from platform presentation at #ASHG15

1. Building a platinum human genome
assembly from single haplotype
human genomes generated from
long molecule sequencing
Karyn Meltz Steinberg
ASHG 2015
@KMS_Meltzy

2. 0
100000
200000
300000
400000
CHM1_1.1 HuRef ALLPATHS YH_2.0
Contig Number
Contig N50
Figure 1
Last year…
Steinberg et al, 2014

3. This year…
0
5000000
10000000
15000000
20000000
25000000
30000000
CHM13
Draft
CHM1
PB_2
CHM1
PB_1
CHM1_1.1 HuRef ALLPATHS YH_2.0
Contig Number
Contig N50

4. This year…
Log
scale
1
10
100
1000
10000
100000
1000000
10000000
100000000
CHM13
Draft
CHM1
PB_2
CHM1
PB_1
CHM1_1.1 HuRef ALLPATHS YH_2.0
Contig Number
Contig N50

5. We combine PacBio with other technologies to construct
the assembly

6. How do we define platinum and gold standards?
GRCh38
Platinum
(CHM1)
Gold
(NA19240)
% Reference genome
covered
100 98.40 90.80
% Assigned chromosomes 99.60 98.40 90.80
% gene models covered
(>95% id, >90% length)
99.96 98.78 94.26
Contig N50 67.8 Mb 26.9 Mb 6.0 Mb
Number of gaps 875 3,640 3,568
Total Assembled size 3.067 Gb 2.996 Gb 2.745 Gb
% haplotype blocks
(>1kb) resolved
NA >95 >80
http://genome.wustl.edu/projects/detail/reference-genomes-improvement/

7. CHM13 Draft Assembly (GCA_000983455.1)
• 60X PacBio (P5 and P6 chemistry)
• Average read length ~11kb
• Daligner/Falcon v 0.2
Total sequence length 2,851,367,788
Number of contigs 2,873
Contig N50 12,981,785
Contig L50 68

8. Gene Model (RefSeq) Analysis
GRCh38
CHM1_
1.1
CHM1_PB1 CHM1_PB2 CHM13
Number of
sequences
not aligning
21 88 67 67 125
Split
Transcripts 8 35 1,245 1,131 285
CDS coverage
<95% 17 266 1,339 1,212 265
Total Sequences Retrieved from Entrez 49,680

9. Short read sequence analysis
• 100X Illumina sequence
• Align with BWA-MEM to ordered and
oriented assembly
• Variant calling via SpeedSeq (Chiang et al,
2015)
• SNVs, indels: FreeBayes
• SVs: LUMPY, SVTyper
• CNV: CNVnator

10. CHM13 Illumina data aligned to CHM13 assembly
202,016 SNVs/indels on unplaced scaffolds
SV_TYPES
>10kb
5-10kb
1-5kb
<1kb
DELETIONS
174
131
430
2582
INVERSIONS
5
0
2
7
DUPLICATIONS
151
112
309
113
TOTAL
330
243
741
2702

11. BioNano SV calls can be used to identify misassembly
Collapse
Expansion
inAssembly
Gap in SequencePacBio Assembly
BioNano Map
SV_TYPES
DELETIONS
41
INVERSIONS
10
INSERTIONS
15
TOTAL
66
BioNano alignment to CHM13

12. BioNano reveals collapse in PacBio assembly
PacBio Assembly
BioNano Map

13. Illumina data aligned to PacBio assembly also shows
collapse

14. BioNano reveals collapse in PacBio assembly due to
highly homologous segmental duplications
SD = 96%
CHR1
46746040
46857004
40
W
LBHZ01000938.1
110965
CHR1
46857005
47034202
41
N
177198
gap
CHR1
47034203
52157695
42
W
LBHZ01000245.1
5123493
PacBio Assembly
BioNano Map

15. This region is rich in medically relevant genes
chr1 (p33) p31.1 1q12 q41 43 44
CYP4Z2P
CYP4A11
CYP4X1
CYP4Z1
CYP4A22
SegDups
Genes
CHM13
PacBio
LBHZ010000938.1 LBHZ010000938.1
LBHZ010000245.1

16. CHM13 Hybrid Scaffold
Hybrid Scaffold
PacBio Contigs
BioNano Contigs

17. CHM13 Hybrid Scaffolds
BioNano Map PacBio Assmbly Hybrid Scaffold
# of Contigs 3593 1590 * 254
Min Contig Length 0.08 Mb 0 0.27 Mb
Median Contig
Length
0.61 Mb 0.06 Mb 4.35 Mb
Mean Contig Length 0.78 Mb 1.78 Mb 9.68 Mb
Contig N50 1.02 Mb 13.46 Mb 20.79 Mb
Max Contig Length 5.27 Mb 63.15 Mb 82.83 Mb
Total Contig Length 2.812 Gb 2.824 Gb 2.458 Gb
*Number of contigs used in hybrid scaffolding

18. Combining CHM1 and CHM13
reference
mapping
CHM1 CHM13
Pipeline analysis
Variant Evaluation
97 13

19. Acknowledgements
The McDonnell Genome Institute at
Washington University in St. Louis
Rick Wilson
Bob Fulton
Wes Warren
Tina Graves-Lindsay
Vince Magrini
Sean McGrath
Derek Albracht
Milinn Kremitzki
Susan Rock
Debbie Scheer
Aye Wollam
The Finishing and Bioinformatics
Teams at The Genome Institute
University of Washington
Evan Eichler
John Huddleston
Archana Raja
NCBI
Valerie Schneider
University of Pittsburgh
School of Medicine (CHM13 cell line)
Urvashi Surti
Personalis
Deanna Church
BioNano Genomics
Palak Sheth
Pacific Biosciences
Jason Chin
Nick Sisneros