This document discusses how linked-read technology from 10x Genomics now enables routine de novo diploid genome assembly from individual human samples. Linked-reads generate long-range genomic information by barcoding DNA fragments derived from the same long input molecules. This allows for improved physical coverage compared to short reads or synthetic long reads. The document shows how a single 10x Genomics library with only 1 ng of input DNA and modest sequencing can generate high-quality human genome assemblies in 2 days. De novo assembly excels at resolving structurally complex and diverse genomic regions that remain difficult for short-read and alignment-based methods.

1. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.© 10x Genomics, Inc. 2016
Everyday de novo diploid assembly
Deanna M. Church
Oct, 2016
@deannachurch

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Disclosures
Employee and Shareholder
Shareholder
10x Genomics
Personalis
10x Genomics products described are for Research Use Only. Not
for use in diagnostic procedures.

3. 3
Acknowledgements
The entire team at 10x
David Jaffe
Neil Weisenfeld
Vijay Kumar
Preyas Shah
Patrick Marks

4. 4
Agenda
•Why haven’t we always done de novo assembly
on every sample?

5. 5
Agenda
•Why haven’t we always done de novo assembly
on every sample?
•What are Linked-Reads?

6. 6
Agenda
•Why haven’t we always done de novo assembly
on every sample?
•What are Linked-Reads?
•What does every day de novo assembly enable today?

7. 7
Why haven’t we always done de novo genome
analysis?

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Current approach: averaging over haplotypes

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Averaging over haplotypes fails with increased diversity
doi:10.1038/nature20098
AK1

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New technology reveals more information

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New technology reveals more information
10.1038/nrg3933

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Public human assemblies to date
https://www.ncbi.nlm.nih.gov/assembly/organism/9606/latest/
Composite genomes Individual Genomes
Hydatidiform moles (single
haplotypes)
• GRCh38
• Celera (2)
• CHM1 (9)
• CHM13 (5)
• NA12878 (9)
• HX1
• A/J Son
• A/J Mother
• A/J Father
• NA18507
• YH1
• HS1011
• AK1
• HuRef
•Lots of labor
•Lots of time
•Lots of coverage
•Lots of money

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What are Linked-Reads?

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Unlinked-Reads: short range information

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Linked-Reads: long range information

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Start with long molecules
NA19240

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Making Linked-Reads
P5 16bp BCR1 Nmer gDNA Insert

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Making Linked-Reads
Long input molecule
Excess of sequenceable
inserts randomly primed
off each long molecule
P5 16bp BCR1 Nmer gDNA Insert

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Making Linked-Reads
Long input molecule (50Kb)
Excess of sequenceable
inserts randomly primed
off each long molecule
P5 16bp BCR1 Nmer gDNA Insert
Long input molecule (50Kb)
30x sequence
~35 fragments
~0.2x coverage
Standard reference based
analysis recommendations

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Making Linked-Reads
Long input molecule (50Kb)
Excess of sequenceable
inserts randomly primed
off each long molecule
P5 16bp BCR1 Nmer gDNA Insert
Long input molecule (50Kb)
56x sequence
~65 fragments
~0.4x coverage
Supernova
analysis recommendations

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Synthetic Long Reads: less physical coverage
CA B
Sequencing cost
Physical coverage

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Linked-Reads: greater physical coverage
CA B
Sequencing cost
Physical coverage

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Linked-Reads allow for increased physical coverage
150X avg physical coverage
Chr13: BRCA2
▲
►
> 56X avg read coverage (assembly)

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Generating Linked-Reads
Start with:
HMW gDNA, 100Kb+ molecules
1.0 ng input DNA = 300 copies of the genome
0.5ng DNA = 150 copies of the genome,
partitioned into > 1M GEMs
DNA
OilBarcoded Primer Library Enzyme Collect

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Assembly made easy
FASTABCL
Supernova
De novo Assembly
1200M
NA19240
http://www.biorxiv.org/content/early/2016/08/19/070425
1 server
348 Gb memory
2 days compute
1 library
1 ng input

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Assembly made easy
FASTABCL
Supernova
De novo Assembly
1200M
NA192401 library
1 ng input
http://www.biorxiv.org/content/early/2016/08/19/070425
1 server (28 cores)
348 Gb memory
2 days compute

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Assembly made easy
FASTABCL
Supernova
De novo Assembly
1200M
NA192401 library
1 ng input
http://www.biorxiv.org/content/early/2016/08/19/070425
1 server (28 cores)
348 Gb memory
2 days compute
megabubble megabubble megabubble

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Performance over multiple human samples
http://www.biorxiv.org/content/early/2016/08/19/070425
sample ethnicity sex cov frag
N50
contig
(Kb)
N50
scaffold
(Mb)
N50
Phase
block
(Mb)
Gap
(%)
NA19238 YRI F 56 115 114.6 18.7 8 2.1
NA19240 YRI F 56 125 118.8 16.4 9.3 2.3
HG00733 PR F 56 106 123.6 17.8 3.4 2.0
HG00512 HAN M 56 102 113.2 15.4 2.7 2.2
NA24385 AJ M 56 120 106.4 15.1 4.2 2.6
HGP EUR M 56 139 120.2 18.6 4.5 2.5
NA12878 EUR F 56 92 118.5 16.4 2.8 2.9

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High quality Assembly at lower coverage
102
104
106
108
110
112
114
116
118
120
122
500 700 900 1,100 1,300
Contig N50 (kb)
Number of reads (millions)
0
5
10
15
20
25
500 700 900 1,100 1,300
Scaffold N50 (Mb)
Number of reads (millions)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
500 700 900 1,100 1,300
Phase Block N50 (Mb)
Number of reads (millions)

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De Novo Performance Drastically Improves with Increased
DNA Length
0
20,000
40,000
60,000
80,000
100,000
120,000
0 10,000 20,000 30,000 40,000 50,000 60,000
Contig N50
0
5
10
15
20
0 10,000 20,000 30,000 40,000 50,000 60,000
Scaffold N50
(Mb)
0
100,000
200,000
300,000
400,000
500,000
0 10,000 20,000 30,000 40,000 50,000 60,000
Phase Block
N50
DNA Length

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Comparison to truth data

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Assembly assessment
Supernova 10x Other methods
0
5
10
15
20
25
NA19238 NA19240 HG00733 HG00512 NA24385 HGP NA12878 YH NA12878 NA12878 NA12878 NA24385 NA24143
Percent GRCh37 100mers missing per assembly
Missing 100mers haploid Missing 100mers diploid
Diploid Haploid

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What does every day de novo assembly
enable?

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Ideal: Complete genome information
doi:10.1038/nature09534
• SNVs
• Deletions
• Insertions
• Inversions
• Translocations

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Areas in which assembly excels: diverse regions
AluY
Supernova
(de novo)
PacBio Reads
Illumina Reads

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Areas in which assembly excels: insertions
Supernova
(de novo)
PacBio
Reads
Illumina
Reads

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Areas in which assembly excels: insertions

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Areas in which assembly excels: insertions
SHANK2
GRCh37: chr11
GRCh37.p13: chr11_fix_patch

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Areas in which assembly excels: insertions
SHANK2
GRCh37: chr11
GRCh37.p13: chr11_fix_patch
35 kb

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Areas in which assembly excels: insertions
Hap1_scaffold 7938
Hap2_scaffold 7939
chr11
SHANK2

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Areas in which assembly excels: insertions
Hap1_scaffold 7938
Hap2_scaffold 7939
chr11
SHANK2

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Areas in which assembly excels: insertions
Hap1_scaffold 7938
Hap1_scaffold 7939
chr11
SHANK2
chr11
Hap2_scaffold 7939
SHANK2
Hap1_scaffold 7938

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Assembly analysis: alignment work needed
SHANK2
Supernova
(de novo)
PacBio Reads
Illumina Reads

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Areas in which assembly excels: inversions
GRCh37 chrX:6137041-6138541 (NLGN4X)
Supernova
(de novo)
PacBio Reads
Illumina Reads

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Assembly analysis: alignment work needed
GRCh37 chrX:6137041-6138541 (NLGN4X)
Hap1_scaffold 5127
Hap2_scaffold 5128

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Fasta is a lossy format
megabubble megabubble megabubble
multi-Mb
phase blocks
many Mb scaffold
micro structure
• bubbles, often at indeterminate poly-A
• short gaps, often at poly-A

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Native formats have more information
Supernova
(de novo)
Long Ranger
Reference
based

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Native formats have more information
Supernova
(de novo)
Long Ranger
Reference
based

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Native formats have more information
Supernova
(de novo)
Long Ranger
Reference
based

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Conclusions
•Routine, de novo, diploid assembly of 1000s of samples
is possible today!
•Early uses will be for better resolution of divergent
regions and novel sequence
•A new generation of tools needs to be developed to
fully utilize assembly data