Click to edit Master title styleClick to edit Master title styleIrys data analysis

January 10th, 2014

Irys Workflow – Data Analysis

Genome Map (.cmap)

Single molecule

maps (.bnx)

Sample Anchoring (.xmap)

irys™ ICS

irysView™

Image processing

Short NGS Contigs

RefSeqReference

Genome Map 2

Structural variation detection

Sequence Assembly Validation

Sequence contig

scaffolding

Integration AnalysisScanning

Sequence scaffolding without de novo assembly

Using a reference (eg hg19)Using a second genome mapUsing NGS contigs

Gross assembly quality (reiterate)Missing sites, extra sites, interval differences structural differences Consed

Alignment in irysviewmanual editingAGP outputConversion to FASTAReimport superscaffolds to reiterate

Mapping based variant calling

Two color applications:

epigenetics, DNA damage

Assembly

workshops

• De novo assembly (Using irysview (Alex); Python/command line – Heng/Ernest)

• SV detection – Warren/Andy

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Core workflow: Data QC: basic molecule stats

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Core workflow: Data QC: molecule quality report

Always consider the mapping rate with respect to the stringency setting

Mapping rate helps us estimate the useful coverage depth as well as data quality

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Stretch normalization

• Evaporation (increasing [salt]) during the scanning prolonged of version 2 chips results in shortening of molecules in nanochannels. This can be corrected for by measuring the average stretch in each scan and correcting with a normalization factor.

• Determining average stretch:– Internal ruler based normalization– Reference mapping based normalization

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Core workflow: De novo assembly: optArg

From molecule quality report and .err file

p value based on genome size or as stringent as possible

Stringencies vary based on step

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No reference?

• With no reference, we can run a de novo assembly based on expectations and data QC observations:– Expected genome size– Site density (in silico)– Label density (empirical)– Molecule n50 (empirical)

• Run de novo assembly (relaxed)• Use the result of the de novo assembly to run molecule

quality report• Update error characteristics (stretch normalization) and rerun

de novo assembly

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De novo assembly QC

We started with 1.8Gb (>100kb) that mapped at 40%. We had a good quality reference so we expect to use ~0.8Gb.

Genome has 14 chromosomes

Expected size is 20Mb

Map n50 is good, we may be able to further improve it with additional depth or optimized sample prep

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De novo assembly QC

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De novo assembly QC

Higher stringency assembly

The higher stringency assembly misses some of the genome but resolves the chimera

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Applications:

Sequence anchoring

12 Mb Streptomyces Genome Assembly with Various Technologies Total Mb Contigs N50 (kb)

9.08 124 92

11.38 97 154

11.63 20 918

11.87 1 11,870

DNA sequence scaffolding

BioNano Genomics

NGS + Cosmids

Short-Read NGS Only

3rd-Gen Reads

Sequence anchoring

Illumina + cosmids: 11.38Mb, 97 contigs, n50 length: 154kb , 11.38Mb anchored

Illumina: 9.08Mb, 124 contigs, n50 length: 92kb , 8.9Mb anchored

Pac Bio: 11.63Mb, 20 contigs, n50 length: 918kb , 11.63Mb anchored

1 Mb

Validate sequence assemblyFind errorsScaffold/Orient/Size gaps

Output FASTA or AGP (soon)

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Applications:

Structural variation

Structural Variation-Insertion/Deletion Calls (vs hg19)

<10 20 30 40 50 60 70 80 90 100

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130

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160

170

180

190

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210

220

230

240

>250

0

10

20

30

40

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60

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Insertion and Deletion size distribution in a normal human genome

Deletion (n=210)

Insertion (n=280)

SV Size (kb)

# of

cal

ls 95 regions in BioNano GenomeMaps correspond to N-based gaps in hg19 (not included in graph). The gaps may contain repeats and polymorphic regions, where SV enriches.

Structural Variant Examples: Insertions and Deletions

Genome Map

hg19M

olec

ules

+4.9kb

Genome Map

hg19

Mol

ecul

es

-176,265 kb#h SmapEntryID QryContigID RefcontigID1 RefcontigID2 QryStartPos QryEndPos RefStartPos RefEndPos Orientation Confidence Type

#f int int int int float float float float string float string net size 1 282 6 6 1,483,278 1,488,217 75,697,428 75,878,632 + -1 delete 176,265

4.9 kb region 181.2 kb region

#h SmapEntryID QryContigID RefcontigID1 RefcontigID2 QryStartPos QryEndPos RefStartPos RefEndPos Orientation Confidence Type#f int int int int float float float float string float string net size

4 577 6 6 1,093,571 1,111,027 13,122,638 13,135,195 + -1 insert 4,899

17.5 kb region 12.6 kb region

workshops

• De novo assembly (Using irysview (Alex); Using Python/command line – Heng/Ernest)– OptArg- iterations, stringencies, merging, ref mapping– Output

• .err file• Alignref• Visualization of genome maps to molecules• Identification of chimeras

• SV detection – Warren/Andy– Explain the SV detection application (consider IP issues)– Discuss stringency parameters– Show resulting table

• ranges• explain types

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