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Poly-T

Long reads and heterozygous SNPs allow you to phase reads into two haplotypes

Candidate gene screening using long-read sequencing

Jenny Ekholm, Ting Hon, Yu-Chih Tsai, David Greenberg, Tyson A. Clark, Steve KujawaPacBio, Menlo Park, CA USA

We have developed several candidate gene

screening applications for both Neuromuscular

and Neurological disorders. The power behind

these applications comes from the use of long-

read sequencing. It allows us to access

previously unresolvable and even unsequencable

genomic regions. SMRT Sequencing offers

uniform coverage, a lack of sequence context

bias, and very high accuracy. In addition, it is also

possible to directly detect epigenetic signatures

and characterize full-length gene transcripts

through assembly-free isoform sequencing.

SMRT Sequencing overview Alzheimer's disease (AD): TOMM40 gene

1) Roses AD, et al. (2010). A TOMM40 variable-length polymorphism predicts the age

of late-onset Alzheimer’s disease. Pharmacogenomics J. 10(5): 375-84

2) Sekar A, et al . (2016). Schizophrenia risk from complex variation of complement

component 4. Nature. 530(7589):177-83

Long-read sequencing data properties

References

Data generated with 20 kb size-selected

human library using 6 hour movies with

P6-C4 chemistry using PacBio RS II,

analyzed with SMRT® Analysis v 2.3.

Each SMRT Cell generates ~55,000.

reads.

E. coli 20 kb-insert library, SMRT

Analysis v2.3

A G C T mA G T T Template strand

C G A G C T AG TTC A T G T Template strand

Example: N6-methyladenine

In addition to calling the bases, SMRT

Sequencing uses the kinetic information from

each nucleotide to distinguish between modified

and native bases.

Informatics Pipeline

Remove adapters

Remove artifacts

Classify

sequence

reads

Reads clustering

Isoform

clusters

Consensus

calling

Nonredundant

transcript

isoforms

Quality

filtering

Final isoforms

PacBio raw

sequence

reads

Map to

reference genome

Experimental pipeline Informatics pipeline

PacBio raw

sequence reads

Figure 1

a b

AAAA

AAAA

AAAAA

AAAAA

AAAAA

AAAAA

AAAAA

Size partitioning &

PCR amplification

cDNA synthesis

with adapters

SMRTbell ligation

RS sequencing

Remove adapters

Remove artifacts

Reads clustering

Quality filtering

Clean

sequence reads

Nonredundant

transcript isoforms

Final isoforms

TTTT

TTTT

Consensus calling

Isoform clusters

Map to reference genome

Evidence-based gene models

polyA mRNA

AAAA

AAAA

TTTT

TTTT

AAAATTTT

AAAATTTT

AAAATTTT

AAAATTTT

Evidenced-based

gene models6 7 8 9 10

DevNet: Iso-Seq wiki page

SampleNet: Iso-Seq Method

with Clonetech cDNA

Synthesis Kit

Raw5’ primer 3’ primer

(AAA)n

(TTT)nSMRT adapter

(TTT)n

(AAA)n

Coding sequencePoly(A) tail

SMRT adapter

(AAA)nReads of Insert(AAA)n

Poly(A) mRNA

AAAAA

AAAAA

AAAAA

AAAAA

cDNA synthesis with

adapters

AAAAATTTTT

AAAAATTTTT

AAAAATTTTT

AAAAATTTTT

AAAAATTTTT

AAAAATTTTT

AAAAATTTTT

AAAAATTTTT

Size Selection & PCR

amplification SMRTbell ligation

Experimental Pipeline

1 2 3 4

Sequel/PacBio RS II

Sequencing

5

A variable poly-T repeat at the rs10524523 SNP

within intron 6 of the TOMM40 gene that in

combination with APOE3 allele will affect the

age of onset of AD1)

The C4 structural variant is highly complex:2)

- Two functionally distinct genes (isotypes); C4A

and C4B

- Both isotypes can have 1 - 3 functional copies

- A human endogenous retroviral (HERV) insertion

in intron 9 changes the length of the gene

Sample 1 All

Sample 1 Phase 0

Sample 1 Phase I

Schizophrenia: C4 gene

Determines if

Long or Short 2 aa difference

in exon 26 determines

C4A or C4B isotype

Different possible

combinations

(L/S/C4A/C4B)

We successfully captured and sequenced the

associated poly-T repeat in the TOMM40 gene

and were able to determine that the sample had

one short (15) allele and one Very Long (34)

allele.

Phase 0

Phase 1 Intron 9

Phase 0

Phase 1

Example: Isotype

C4A/L was

successfully

called for a

sample using

long-read

sequencing

Gene isoform characterization

Amplification-free targeted sequencing

using CRISPR/Cas9

Repeat expansion disorders are challenging to

interrogate due to the long repetitive regions.

Using CRISRP/Cas9 we are able to access the

repeat counts, interruption sequences as well as

epigenetic information without introducing PCR

bias.

Targeted sequencing and multiplexing

Targeted sequencing workflow Barcoding options

2. Barcoded Universal Primer

1. Barcoded adapters

C4 plays a role in signaling which connections

between neurons should be “pruned” or removed,

as the brain develops after childhood. And the

more C4 was present, the higher the risk of

developing schizophrenia. Certain versions of the

C4 gene seem to increase people’s risk for

developing schizophrenia by 27 to 50 percent.

We successfully captured and sequenced the

C4 gene as part of a MHC capture panel. We

were able to see the two different C4 isotypes

(C4A and C4B) as well as seeing the 7 kb

HERV insertion in intron 9.

The Huntingtin gene

CAG repeat counts in

HD patients

Direct methylation detection of FMR1

pre-mutation sample

CGG repeat region appears to be heavily

methylated (5mC)

HTT gene in HD sample

gDNA & Transcripts from SK-BR-3 Cell Line Captured with NimbleGen

Oncology Panel - example AURKA gene

Phased Transcripts reveal retained introns and skipped exons

Read Length Accuracy