101717.kh miga ashg_grc

Centromere Sequence Assembly

Karen H. Miga University of California, Santa Cruz

10/17/17GRC GIAB Workshop

ASHG

Megabase-sized gapsP-ARM Q-ARM

CEN

HUMAN CENTROMERES: MULTI-‐MEGABASE SIZED GAPS IN ALL CHROMOSOME ASSEMBLIES

PROGRESS UPDATE: CENTROMERE SEQUENCE ASSEMBLIES

1. GRCh38 Reference Models for Human Centromere Arrays

2. Efforts to Generate True, Linear Assemblies of Centromeric regions: Chromosome Y

3. Future PerspecSve

p-arm q-arm... ...Multi-megabase sized arrays of satellite DNA

...ATCCGATTACG ATCCGATTACGATCCGATTACG... ...ATCCGATTACG ATCCGATTACGATCCGATTACG...

CHALLENGE OF ASSEMBLING LONG TRACTS OF (NEAR IDENTICAL) TANDEM REPEATS

p-arm q-arm... ...ALPHA SATELLITE

~171bp Tandem Repeat

Wide Range of Percent ID: ~60-100%

1 2 3 4

HUMAN CENTROMERES: ALPHA SATELLITTE

Narrow Range of Percent ID: 94% - 100%

“Higher Order Repeat”

Multi-monomeric Repeat Unit

p-arm q-arm... ...

1 2 3 4 1 2 3 4 1 2 3 4

HIGHER ORDER REPEATS

p-arm q-arm... ...

p-arm q-arm... ...

Array “A”

Array “B” Array “C”

chrX

chr3

CHROMOSOME-‐SPECIFIC SATELLITE SEQUENCE ORGANIZATION

p-arm q-arm... ...

... ...-A- -T-

GENOME MODEL OF SEQUENCE ORGANIZATION IN CENTROMERE-‐ASSIGNED GAPS

p-arm q-arm... ...

... ...-A- -T-


LINESINE

OTHER

NON-ALPHA SATELLITE

p-arm q-arm... ...

... ...-A- -T-


LINESINE

OTHER

NON-ALPHA SATELLITE

Unmapped (Yet Assembled) Scaffolds

Characterize HORs in Human Genome1

1. GRCh38 Alpha Satellite Reference Models

1

A B C D E F



1

>200 ENCODE datasets

A B C D E F



>200 ENCODE datasetsStep by Step Example For Single P-read, I

•  Example: p-read 78e9dc60_12326_5223 (internal read identifier) –  p-read length 12257

–  Self-self dot plot

22

0 2000 4000 6000 8000 10000 12000

2000

4000

6000

8000

10000

12000

0

α-Centauri (centromeric automated repeat identification)

12,257 bp PacBio Read with 70 adjacent monomers (NA12878, 10x Coverage HuPac Data)

5’…

…3’

10x

10

B

C

D

E

F

A10

10

1010

10

5’ 3’

1

http://github.com/volkansevim/alpha-CENTAURI.

B

C

D

E

F

A

Chromosome specific assignment

?

Experimental Evidence: FISH Hybridization/Mapping and Screening Somatic Cell Hybrid Panel

B

C

D

E

F

A

D7Z16-mer

Waye et al (1987) 98% GenBank: M16101

Flow Sorted Chromosome Alignment/Enrichment Sequence enrichment analysis of isolated human chromosomes

Long Range Paired Read Support“Anchor” to mapped to the assembled p-arm and/or q-arm

Chromosome specific assignment

Chromosome-assignment of Higher Order Repeats

Characterize HORs in Human Genome


DXZ1 (12-mer)CENX

e.g.1 2 3 4 5 6 7 8 9 10 11 12

LINEHuRef WGS Sanger

read Db

Constructing WGS Read Libraries for each HOR array2

HuRef

LINEA/T

1

Characterize HORs in Human Genome


Constructing WGS Read Libraries for each HOR array

m3v1

m1v1m2v1

m2v2

m4v1

m12v1

m5v1

m6v1

LINE

m11v1

m10v1

m9v1

m8v1

m7v1

1.01.0

1.0

1.0

1.0

1.0

1.0

0.5

0.5

0.5

0.3

0.70.3

0.7

1.0

LINEA/T

2

1

3 Model Array Variants in Sequence Graph:

linearSat• 2nd Order Markov Chain• Length determined by normalized array length estimates

m3v1

m1v1m2v1

m2v2

m4v1

m12v1

m5v1

m6v1

LINE

m11v1

m10v1

m9v1

m8v1

m7v1

1.01.0

1.0

1.0

1.0

1.0

1.0

0.5

0.5

0.5

0.3

0.70.3

0.7

1.0

Not the “true” long-range organization, yet adequately represents the alpha satellite

array sequence

https://github.com/JimKent/linearSat

https://github.com/JimKent/linearSat

LINEAR ORDERING OF REFERENCE MODELS AND ASSEMBLED CONTIGS USING MATE PAIRS

CENXXp Xq

3.8 Mb

chrX

2.25Mb; ~860 HOR units 0.73Mb; ~43 HOR units

0.3Mb; Low Copy Repeat

pp3p 3qCEN3.1 CEN3.2Unmapped HuRef

Assembled Contig(s)(e.g. ABBA01185959)

chr3

Yp Yq

Xp Xq

100Kb

12p 12q

17q17p

2p 2q

6p 6q

3p 3q

4p 4q

11p 11q

8p 8q

10p 10q

7p 7pq 7q

9q9p

1p 1q

16q16p

18p 18q

19p 19q

20q20p

5p 5q

1

2

3

4

5

6

7

8

9

10

11

12

15

16

17

18

19

20

15q 15p

X

Y

21p14q

21q

Acrocentric Chr (13,14,21,22)

An Initial Draft of Human Centromere

Sequence Composition

Alpha Satellite Reference Models: ~60 Mb (59571670 bp)

CENTROMERE SEQUENCE ASSEMBLY


2. Linear Assembly of a Human Centromere Miga, KH., et al. Genome research 24.4 (2014): 697-707.l 20

LINEAR ASSEMBLY OF A HUMAN CENTROMERE ON THE Y CHROMOSOME

Small, haploid satellite array with well-characterized 5.8 kb repeat

p-arm q-arm

BACS: OVERLAP-‐LAYOUT-‐ASSEMBLY

p-arm q-arm

Collection of 9 BACs known to span the Y Centromere

Overlap determined by single copy sequence variants

Tilford et al 2001 Nature

HIGH QUALITY + LONG (100 kb +) READS

~100 kb

Collapsed Representation

Challenge of Assembling

Identical Tandem Repeats with Short

Reads

HIGH QUALITY + LONG (100 kb +) READS

High Quality Consensus Sequence

~100 kb

NANOPORE SEQUENCING: LONGBOARD (1D)UCSC LONGBOARD 1D PROTOCOL

UCSC LONGBOARD 1D PROTOCOL NANOPORE SEQUENCING: LONGBOARD (1D)

UCSC LONGBOARD 1D PROTOCOL

In total, we have generated 3500+ reads greater than 150 kb

NANOPORE SEQUENCING: LONGBOARD (1D)

MULTIPLE ALIGNMENT STRATEGY TO IMPROVE QUALITY BY CONSENSUS

High Quality Consensus Requires

Modest Coverage

UCSC LONGBOARD 1D PROTOCOL MULTIPLE ALIGNMENT STRATEGY TO IMPROVE QUALITY BY CONSENSUS

RP11 718M18 221.4 kb

Vector

Insert

634 Predicted Nucleotide Variants

2 Tandem Structural Rearrangements

38 CENY RPTS (>99% Identity to published consensus)

Homopolymers [A]n

Homopolymers [T]n

Identify informative, single copy sites in the array useful for overlap BAC-based assembly

IDENTIFY SINGLE COPY VARIANTS USING ILLUMINA DATA

RP11 718M18 221.4 kb

VALIDATE HIGH-‐CONFIDENT SINGLE COPY VARIANTS WITH ILLUMINA

RP11 718M18 221.4 kb

VALIDATE HIGH-‐CONFIDENT SINGLE COPY VARIANTS

LINEAR ASSEMBLY OF HUMAN Y CENTROMERE

Future PerspecSve

1. Linear assemblies of human centromeric regions improve in step with sequencing technology (i.e. read length and quality)

2. One genome is not enough: Highly variable

3. Linear CEN assemblies present a mapping challenge to most genomic applicaSons

True Linear Maps of Human CEN Regions

Y CEN

True Linear Arrangement

Informatics/Analysis Data Structure

Key Advantages of Satellite DNA Graphs

1. Eliminates sequence redundancy


Improves Unambiguous Short Read Mapping

REPEAT REPEAT REPEAT

?

5’ 3’REPEAT

Benedict Paten Adam Novak

Centromere GraphsDemonstrate unambiguous mapping the majority ( > 98%) of 1000 genome alpha satellite reads




2. Information describing long-range haplotypes are retained as defined “paths” in the graph:



2. Information describing long-range haplotypes are retained as defined “paths” in the graph

3. Graph data structure and sequence analysis tools will be consistent with the rest of the human genome

The major histocompatibility complex (Kiran Garimella & Gil McVean)

Creating (and mapping to) a Universal Reference Genome

Benedict Paten, Adam Novak, David Haussler, UC Santa Cruz

Mark Akeson Miten Jain Hugh Olsen Benedict Paten Dave Deamer

Robin AbuShumays Andrew Smith Ian Fiddes Art Rand Logan Mulroney

Jordan Eizenga Rojin Safavi Rachel Lawton Andrew Bailey Ariah Mackie

David HausslerBenedict Paten

Jim Kent Sofie Salama

UCSC Nanopore Analysis GroupMiten Jain Hugh Olsen Mark Akeson

Dan TurnerDavid Stoddart

Oxford Nanopore Technologies

Huntington F. WillardDavid Page

Product Version

Device MinION MK1

Flow cell FLO-MIN106

Kits Rapid Sequencing Kit

Data analysis

Albacore 1.0.1 Metrichor 1D

Acknowledgements

101717.kh miga ashg_grc

Health & Medicine