Presentation by Karen Miga at GRC/GIAB ASHG 2017 workshop "Getting the most from the reference assembly and reference materials" on centromere assemblies.

1. Centromere Sequence Assembly
Karen H. Miga
University of California, Santa Cruz
10/17/17
GRC GIAB Workshop
ASHG

2. Megabase-sized gapsP-ARM Q-ARM
CEN
HUMAN
CENTROMERES:
MULTI-­‐MEGABASE
SIZED
GAPS
IN
ALL
CHROMOSOME
ASSEMBLIES

3. CEN

4. 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

5. 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

6. p-arm q-arm
... ...ALPHA SATELLITE
~171bp
Tandem Repeat
Wide Range of Percent ID: ~60-100%
1 2 3 4
HUMAN
CENTROMERES:
ALPHA
SATELLITTE

7. 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

8. p-arm q-arm
... ...
p-arm q-arm
... ...
Array “A”
Array “B” Array “C”
chrX
chr3
CHROMOSOME-­‐SPECIFIC
SATELLITE
SEQUENCE
ORGANIZATION

9. p-arm q-arm
... ...
... ...-A- -T-
GENOME
MODEL
OF
SEQUENCE
ORGANIZATION
IN
CENTROMERE-­‐ASSIGNED
GAPS

10. p-arm q-arm
... ...
... ...-A- -T-
GENOME
MODEL
OF
SEQUENCE
ORGANIZATION
IN
CENTROMERE-­‐ASSIGNED
GAPS
LINE
SINE
OTHER
NON-ALPHA SATELLITE

11. p-arm q-arm
... ...
... ...-A- -T-
GENOME
MODEL
OF
SEQUENCE
ORGANIZATION
IN
CENTROMERE-­‐ASSIGNED
GAPS
LINE
SINE
OTHER
NON-ALPHA SATELLITE
Unmapped
(Yet Assembled) Scaffolds

12. Characterize HORs in Human Genome1
1. GRCh38
Alpha
Satellite
Reference
Models
1

13. A B C D E F
Characterize HORs in Human Genome1
1. GRCh38
Alpha
Satellite
Reference
Models
1

14. >200 ENCODE datasets
A B C D E F
Characterize HORs in Human Genome1
1. GRCh38
Alpha
Satellite
Reference
Models
>200 ENCODE datasets
y Step Example For Single P-read, I
α-Centauri
(centromeric automated repeat identification)
5’…
…3’
10x
10
B
C
D
EF
A
10
10
10
10
10
5’ 3’
1
http://github.com/volkansevim/alpha-
CENTAURI.

15. B
C
D
EF
A
Chromosome specific assignment
?

16. Experimental Evidence:
FISH Hybridization/Mapping and Screening Somatic
Cell Hybrid Panel
B
C
D
EF
A
D7Z1
6-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

17. Chromosome-assignment of Higher Order Repeats

18. Characterize HORs in Human Genome
1. GRCh38
Alpha
Satellite
Reference
Models
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
LINEA/T
1

19. Characterize HORs in Human Genome
1. GRCh38
Alpha
Satellite
Reference
Models
Constructing WGS Read Libraries for each HOR array
m3v1
m1v1
m2v1
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
0.7
0.3
0.7
1.0
LINEA/T
2
1
3 Model ArrayVariants in Sequence Graph:

20. linearSat
• 2nd Order Markov Chain
• Length determined by normalized
array length estimates
m3v1
m1v1
m2v1
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
0.7
0.3
0.7
1.0
Not the “true” long-range
organization, yet adequately
represents the alpha satellite
array sequence
https://github.com/JimKent/linearSat

21. LINEAR
ORDERING
OF
REFERENCE
MODELS
AND
ASSEMBLED
CONTIGS
USING
MATE
PAIRS
CENXXp Xq
3.8 Mb
chrX
2.25Mb; ~860 HOR units0.73Mb; ~43 HOR units
0.3Mb;
Low Copy
Repeat
pp
3p 3qCEN3.1 CEN3.2
Unmapped HuRef
Assembled Contig(s)
(e.g.ABBA01185959)
chr3

22. 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
21p
14q
21q
Acrocentric Chr
(13,14,21,22)
An Initial Draft of
Human Centromere
Sequence Composition
Alpha
Satellite
Reference
Models:
~60
Mb
(59571670
bp)

23. CENTROMERE
SEQUENCE
ASSEMBLY
1. GRCh38
Alpha
Satellite
Reference
Models
2. Linear
Assembly
of
a
Human
Centromere
Miga, KH., et al. Genome research 24.4 (2014): 697-707.l 20

24. 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

25. 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

26. HIGH
QUALITY
+
LONG
(100
kb
+)
READS
~100 kb
Collapsed
Representation
Challenge of
Assembling
Identical Tandem
Repeats with Short
Reads

27. HIGH
QUALITY
+
LONG
(100
kb
+)
READS
High Quality Consensus Sequence
~100 kb

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

29. LONGBOARD 1D PROTOCOL
NANOPORE
SEQUENCING:
LONGBOARD
(1D)

30. UCSC LONGBOARD 1D PROTOCOL
In total, we have generated 3500+ reads
greater than 150 kb
NANOPORE
SEQUENCING:
LONGBOARD
(1D)

31. MULTIPLE ALIGNMENT STRATEGY TO IMPROV
QUALITY BY CONSENSUS
High Qualit
Consensus Req
Modest Cove
UCSC LONGBOARD 1D PROTOCOL
MULTIPLE
ALIGNMENT
STRATEGY
TO
IMPROVE
QUALITY
BY
CONSENSUS

32. 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

33. Identify informative, single
copy sites in the array useful
for overlap BAC-based
assembly
Y SINGLE COPY VARIANTS USING ILLUMINA DATA
RP11 718M18
221.4 kb
VALIDATE
HIGH-­‐CONFIDENT
SINGLE
COPY
VARIANTS
WITH
ILLUMINA
RP11 718M18
221.4 kb

34. VALIDATE
HIGH-­‐CONFIDENT
SINGLE
COPY
VARIANTS

35. LINEAR
ASSEMBLY
OF
HUMAN
Y
CENTROMERE

36. 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

37. True Linear Maps of Human CEN Regions
Y CEN
True Linear
Arrangement
Informatics/Analysis
Data Structure

38. Key Advantages of Satellite DNA Graphs
1. Eliminates sequence redundancy

39. Key Advantages of Satellite DNA Graphs
Improves Unambiguous Short Read Mapping
REPEAT REPEAT REPEAT
?
5’ 3’REPEAT
Benedict Paten Adam Novak
Centromere Graphs
Demonstrate unambiguous mapping
the majority ( > 98%) of
1000 genome alpha satellite reads
1. Eliminates sequence redundancy

40. Key Advantages of Satellite DNA Graphs
1. Eliminates sequence redundancy
2. Information describing long-range haplotypes are
retained as defined “paths” in the graph:

41. Key Advantages of Satellite DNA Graphs
1. Eliminates sequence redundancy
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)

42. 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 Haussler
Benedict Paten
Jim Kent
Sofie Salama
UCSC Nanopore Analysis Group
Miten Jain Hugh Olsen Mark Akeson
Dan Turner
David Stoddart
Oxford Nanopore Technologies
Huntington F. Willard
David Page
Product Version
Device MinION MK1
Flow cell FLO-MIN106
Kits Rapid Sequencing Kit
Data
analysis
Albacore 1.0.1
Metrichor 1D
Acknowledgements