This document describes a method for haplotype-resolved structural variant assembly using long reads. PacBio and BioNano data are hybrid assembled to generate highly contiguous and complete haplotype-specific assemblies. The hybrid approach resolves many gaps in current references assemblies and detects complex structural variants and rearrangements. Analysis of trios from the 1000 Genomes Project and GIAB project using this pipeline detects numerous insertions, deletions, inversions and other structural variants.

1. Haplotype resolved structural
varia1on assembly with long reads
Mount Sinai: Ali Bashir, Oscar
Rodriguez, MaAhew Pendleton
Reed College: Anna Ritz, Alex Ledger

2. Overview
• Background
– Automated Hybrid Assembly
• Phased Diploid Assembly
– Exisi1ng Limita1on of assembly
– 10X + PacBio
• Issues with calling SVs and comparing datasets

3. Pacbio: 44X, ~4.9 kb avg. length BioNano: 80X, 278kb mean span
Technologies of Greater Scale Needed
to Study Human Complexity

4. Sequence Contigs
Contig Maps
In silica diges1on
Genome Maps
Aligned Contig-Scaffold Pairs
NGS
De Novo Assemble
Align Genome Maps to Con6gs /
Con6gs to Genome Maps
Scaffold Graph Construc6on and
Layout
A. Pang M. Pendleton
BioNano Raw Molecules
Hybrid Scaffolds
Schema1c for Hybrid Scaffolding

5. Sequence Contigs
Contig Maps
In silica diges1on
Genome Maps
Aligned Contig-Scaffold Pairs
NGS
De Novo Assemble
Align Genome Maps to Con6gs /
Con6gs to Genome Maps
Scaffold Graph Construc6on and
Layout
A P.ang M. Pendleton
BioNano Raw Molecules
Hybrid Scaffolds
Scaffolding is symmetric
Schema1c for Hybrid Scaffolding

6. Sequence Contigs
Contig Maps
In silica diges1on
Genome Maps
Aligned Contig-Scaffold Pairs
NGS
De Novo Assemble
Align Genome Maps to Con6gs /
Con6gs to Genome Maps
Scaffold Graph Construc6on and
Layout
A P.ang M. Pendleton
BioNano Raw Molecules
Hybrid Scaffolds
Inconsistencies can
be flagged to break or
eliminate ConHgs or
Genome Maps
Schema1c for Hybrid Scaffolding

7. As anHcipated, heterochromaHc regions are most difficult to span
-Completes as many as 28 gaps in hg38
-Other methods and data types can be used to further resolve addiHonal gaps in the assembly
Hybrid Assembly Between BioNano & PacBio Supercon1gs

8. Hybrid Assembly Between BioNano & PacBio Supercon1gs
As anHcipated, heterochromaHc regions are most difficult to span
-Completes as many as 28 gaps in hg38
-Other methods and data types can be used to further resolve addiHonal gaps in the assembly
Orthogonal Error Profiles enable drama1c improvements

9. hg19
chr1
Super
scaffold
hg38
chr1
Hg19 gap Hg19 gap
Super
scaffold
Molecule
pileup
Complex Structural Rearrangements can be Validated
Rela1ve to Current References
S1ll resolves at least
28 gaps in hg38
assembly for >400kb
in predicted gap
intervals
Many large structural variants predicted
like the above.
Are they real?

10. hg19
chr1
Super
scaffold
hg38
chr1
Hg19 gap Hg19 gap
Super
scaffold
Molecule
pileup
Complex Structural Rearrangements can be Validated
Rela1ve to Current References
S1ll resolves at least
28 gaps in hg38
assembly for >400kb
in predicted gap
intervals

11. Very Large TR Expansions Detected via Op1mal Map
LPA (Kringle IV Exon Expansion – Each Period >5kb)

12. Example Signatures of Complex Events

13. Most Inversions From the 1000 Genomes
Project Are Not Actually Inversions!

14. • GIAB
– Long-read sequencing of Trios
• 20-30X parents
• 40-70X children
– 10X Chromium Data
– Ashkenazi Jewish
• IkG
– Long-read sequencing
• ~20-25X parents
• ~45-50X parents
– 10X Chromium Data
• Chinese, Puerto Rican, and Yorbuan
ancestry
Technology Con1nues to March Forward
GIAB AJ and 1000 Genomes Trios
Sub RL N50 = 11,087 bp
Total # Bases = 220 Gb
# of Reads = 27.4 M reads

15. Current Genome Assembly Results on GIAB and 1kG Trios
Sample Contigs Average N50 Max Total Size
HG002 13231 230 kb 4.1 Mb 31.6 Mb 3.04 Gb
HG003 17873 172 kb 4.6 Mb 21.5 Mb 3.08 Gb
HG004 16487 185 kb 5.3 Mb 22.6 Mb 3.05 Gb
HG00512 23146 117 kb 369 kb 2.6 Mb 2.72 Gb
HG00513 18443 151 kb 401 kb 2.4 Mb 2.78 Gb
HG00514 11517 264kb 7.2 Mb 61.1Mb 3.04Gb
HG00731 20811 132 kb 451 kb 3.8 Mb 2.74 Gb
HG00732 13672 214 kb 1.3 Mb 10.9 Mb 2.93 Gb
HG00733 11143 281kb 11.4 Mb 57.4 Mb 3.14 Gb
NA19238 56480 39 kb 70 kb 645 kb 2.20 Gb
NA19239 73478 23 kb 40 kb 1.01Mb 1.71 Gb
NA19240 15245 203 kb 3.8 Mb 20.1 Mb 3.09 Gb
Joyce Lee (BioNano Genomics)
Sample Enzyme Contigs N50 Total Size
HG00733 (Genome Map) BspQI 2185 4.2 Mb 5.6 Gb
HG00733 (Genome Map) BssSI 5038 1.39 Mb 5.1 Gb
HG00733 (Hybrid) BspQI 133 / 10637 56.4 Mb / 52.2 Mb 2.8 Gb
HG00733 (Hybrid) BssSI 234 / 10749 40.26 Mb / 29.7 Mb 2.8 / 3.2 Gb
HG00733 (Hybrid) BspQI + BssSI 104 / 10590 72.6 Mb / 61.5 Mb 2.9 Gb / 3.2 Gb
Hybrid Assembly with mul1ple Enzymes drama1cally
improves con1guity and coverage of the genome

16. Current Genome Assembly Results on GIAB and 1kG Trios
Sample Contigs Average N50 Max Total Size
HG002 13231 230 kb 4.1 Mb 31.6 Mb 3.04 Gb
HG003 17873 172 kb 4.6 Mb 21.5 Mb 3.08 Gb
HG004 16487 185 kb 5.3 Mb 22.6 Mb 3.05 Gb
HG00512 23146 117 kb 369 kb 2.6 Mb 2.72 Gb
HG00513 18443 151 kb 401 kb 2.4 Mb 2.78 Gb
HG00514 11517 264kb 7.2 Mb 61.1Mb 3.04Gb
HG00731 20811 132 kb 451 kb 3.8 Mb 2.74 Gb
HG00732 13672 214 kb 1.3 Mb 10.9 Mb 2.93 Gb
HG00733 11143 281kb 11.4 Mb 57.4 Mb 3.14 Gb
NA19238 56480 39 kb 70 kb 645 kb 2.20 Gb
NA19239 73478 23 kb 40 kb 1.01Mb 1.71 Gb
NA19240 15245 203 kb 3.8 Mb 20.1 Mb 3.09 Gb
Joyce Lee (BioNano Genomics)
Hybrid Assembly with mul1ple Enzymes drama1cally
improves con1guity and coverage of the genome
Sample Enzyme Contigs N50 Total Size
HG00733 (Genome Map) BspQI 2185 4.2 Mb 5.6 Gb
HG00733 (Genome Map) BssSI 5038 1.39 Mb 5.1 Gb
HG00733 (Hybrid) BspQI 133 / 10637 56.4 Mb / 52.2 Mb 2.8 Gb
HG00733 (Hybrid) BssSI 234 / 10749 40.26 Mb / 29.7 Mb 2.8 / 3.2 Gb
HG00733 (Hybrid) BspQI + BssSI 104 / 10590 72.6 Mb / 61.5 Mb 2.9 Gb / 3.2 Gb

17. Previous Work in NA12878:
Hybrid Assembly/Varia1on Analysis Pipeline
Pendleton et al., Nature Methods 2010
M. Pendleton
A. Pang
J. Chin

18. Previous Work in NA12878:
Hybrid Assembly/Varia1on Analysis Pipeline
Pendleton et al., Nature Methods 2010
M. Pendleton
A. Pang
J. Chin

19. Previous Work in NA12878:
Hybrid Assembly/Varia1on Analysis Pipeline
Pendleton et al., Nature Methods 2010
M. Pendleton
A. Pang
J. Chin

20. Summary Of 1kG/GIAB PacBio SV Calls
*Ran through a streamlined version of the SV pipeline may not be comparable
Other Notes:
- MEI calls use conserva1ve parameters (likely undercalling inser1ons)
- “Other” calls contain some improperly flagged inser1ons/dele1ons as well as complex
events and inversions
InserHon DeleHon Complex
Sample
# of
Calls
# of TR
calls
# of
Alu
#
of L1
# of
SVA # of Calls
# of TR
calls*
# of
Alu # of L1 # of SVA
HG002 13471 5573 325 68 7 9639 6880 798 201 22 2493
HG003* 12947 5133 411 74 5 9692 6776 411 74 5 2580
HG004 12769 5066 475 160 96 9509 7233 971 282 33 2599
HG00512 9830 4164 366 75 67 7672 5781 768 275 23 2157
HG00513 9761 4175 351 86 79 7791 5936 770 258 27 2314
HG00514 1285 4866 212 42 3 9636 6770 767 222 26 2635
HG00731 9874 4322 357 76 75 7678 5797 790 256 17 2174
HG00732 11059 4884 400 85 85 8227 6274 813 271 24 2351
HG00733* 11769 5365 330 45 4 8848 6179 743 191 25 2313
NA19238 7512 2999 280 72 59 6320 4765 628 237 12 1910
NA19239 5909 2357 199 46 50 5061 3809 528 161 21 1468
NA19240* 13285 5185 345 78 7 9791 7596 911 275 23 2600

21. Venn Diagrams Between all Trios:
YRI/PUR/CHS Dele1ons
Not seeing all proband calls (blue) in the parents
- Under-calling of hets?

22. Haplotype Resolved Assembly
Par11oned assembly of PacBio reads with
10X Genomics phased variant calls

23. Assembly Sequences Oven Mix
Haplotype Informa1on
hAp://support.10xgenomics.com/de-novo-assembly/sovware/pipelines/latest/output/genera1ng
Bubbles represent divergent alleles
Blue = Maternal
Yellow = Paternal

24. hAp://support.10xgenomics.com/de-novo-assembly/sovware/pipelines/latest/output/genera1ng
Bubbles represent divergent alleles
Blue = Maternal
Yellow = Paternal
A conserva1ve assembly will NOT try to
link across the black bubbles without
some sort of scaffolding informa1n
Assembly Sequences Oven Mix
Haplotype Informa1on

25. hAp://support.10xgenomics.com/de-novo-assembly/sovware/pipelines/latest/output/genera1ng
Bubbles represent divergent alleles
Blue = Maternal
Yellow = Paternal
Assemblies oven take a single path in this graph,
this could mix maternal and paternal alleles
Assembly Sequences Oven Mix
Haplotype Informa1on

26. 10X + PacBio: Haplotype Resolved SVs
Genome
10X par11oned SNVs
Long
reads

27. Overview of Approach

28. Overview of Approach

29. Overview of Approach

30. Revised Call Sets
* PacBio calls take from “Sniffles” – developed at Schatz Lab (John Hopkins)
** 10X calls take provided using LongRanger from 10X Genomics

31. Hybrid Haplotype Separated Callsets Add
Many SVs
* PacBio calls take from “Sniffles” – developed at Schatz Lab (John Hopkins)
** 10X calls take provided using LongRanger from 10X Genomics

32. Combined Calls Largely Contain
Exis1ng Calls
(Sniffles SV Detec1on)

33. Calls Remain That Are Unique To A
Single Plaxorm
(Sniffles SV Detec1on)

34. Assembly Calls “Missed” In Haplotype Calls
Par11oned
Reads
Haplotype
Con1gs
Assembly Calls
Haplotype Calls

35. Haplotype Calls “Missed” In Assembly Calls
Par11oned
Reads
Haplotype
Con1gs
Assembly Calls
Haplotype Calls

36. Haplotype Calls “Missed” In Assembly Calls
Par11oned
Reads
Haplotype
Con1gs
Assembly Calls
Haplotype Calls

37. Repeats (like TRs) can shiv
boundaries for various callers

38. Example Workarounds
-Breakpoint calls (not assembled sequence):
If L1 is within 10% of L2 size and L1 and L2 are both in R
we call it a “match”
- Assembly Calls
Perform MSA to iden1fy truly “homozygous”
sequences
L1
L2
Tandem Repeat

39. Complex hets Are Oven in “Hard regions”
Par11oned
Reads
Haplotype
Con1gs
Assembly Calls
Haplotype Calls

40. Assembly Provides Detailed
Indica1ons Of Quality
• Provides sequence of breakpoint
• Poten1ally provides co-located events
• Poten1ally provides informa1on on accuracy
of the assembly in that region

41. Slide from Jason Chin
at the SMRT
Informa6cs Workshop
Ctg 33
Ctg33mappedtoChr1
Ctg 120
Mis-assembly point
Assembly have addi1onal informa1on

42. Ongoing/Future Work
• GIAB is working on how to integrate assembly calls robustly
– Surprisingly poor overlap!
• Typing calls
• Integra1ng parental informa1on
• Providing full length haplotype assemblies for genomes
– Can be done with trio phasing
– But, it’s now possible with new technologies!
• Hi-C
• StrandSeq
• Integra1ng Graphs from 10x and PacBio
• Pulling in very large SVs with BioNano
• Moving into full indel resolu1on and tools for comparing datasets
rapidly to alt haps

43. Acknowledgements
• Mount Sinai
– Eric Schadt
– Matt Pendleton
– Ajay Ummat
– Oscar Franzen
– Gintaras Deikus
– Robert Sebra
– Oscar Rodriguez*
• Reed College
– Anna Ritz
– Alex Ledger
• UCSF
– Pui Kwok
• PacBio
– Jason Chin
• 1000 Genomes SV
Working Group
• UW
– Mark Chaisson
• EMBL
– Jan Korbel
– Markus H.-Y.
Fritz
– Tobias Rausch
• BioNano Genomics
– Han Cao
– Alex Hastie
– Heng Dai
– Andy Pang
– Joyce Lee
• 10X Genomics
– Patrick Marks
– Deanna Church
– Mike Schnall-Levin
– Sofia
Kyriazopoulou-
Panagiotopoulou