Piloting an approach to cloning and sequencing genome rearrangements using follicular lymphoma as a case study.

A strategy for DNA sequence analysis of genome rearrangements

Background

“Low resolution, genome-wide studies are beginning to catalog additional changes, such as small deletions or amplifications, and directed studies of individual genes implicated in tumor biology are steadily increasing our awareness of the variety of mutations underlying cancer. These data make it evident that only a modest fraction of the molecular targets involved in tumorigenesis has been identified and that cancer is a very heterogeneous disease due to many different mutations, environmental factors, and the interaction between the two. To develop targeted interventions, it will be important to identify all or most of these events.”

“These technological improvements make obtaining sequence information increasingly rapid and relatively inexpensive. It is now possible to contemplate something that was previously incomprehensible: obtaining comprehensive sequence information from multiple tumor types, at different stages—in a process that would be unbiased by our currently selective knowledge of the biology of cancer—to catalog all the genomic changes associated with cancer, and to render them accessible to study and intervention.”

Exploring Cancer through Genomic Sequence Comparisons. A NCI – NHGRI Workshop, April 14-15, 2004.

Charge to the workshop

• Which sequencing technologies would be the most appropriate for this application?

• Which tumor(s) should receive initial focus?

• What other data should be collected on selected tumor types?

• How could such an effort be piloted?

Sequencing genome rearrangements could provide a perspective on what could be learned from whole

genome sequencing of cancers.

How can these be found (and sequenced)?

What cancers?

What stages of cancer?

RFI: Large-scale identification of somatic mutations in cancer

Genomic Instability and Disease

genomic rearrangements play a major role in pathogenesis of human genetic disease architectural features of the genome are associated with susceptibility to rearrangements segmental duplications are implicated in facilitating recombination events that lead to

rearrangements rearrangements are not strictly random events but reflect higher order genome architectural features

alteration of gene dosage or creation of novel fusion genes as a result of recombination red-green colour blindness is caused by frequent deletions/duplications between highly

similar red and green opsin loci (Xq28)Stankiewicz, P. and Lupski, J.R. 2002. Molecular-evolutionary mechanisms for genomic disorders. Curr Opin Genet Dev 12: 312-319.

Lymphoma and Leukemia-associated Rearrangements

Janz, S. et al. 2003. Genes Chromosomes Cancer 36: 211-223.

Follicular lymphoma as a test case

The incidence of Follicular Lymphoma in Canada is significant and increasing. New approaches for disease control are required, and these will most likely derive from enhanced understanding of disease biology.

There is a strong lymphoma research group at BCCA and UBC.

FL samples of > 90% “purity” can be obtained in quantities sufficient for BAC library construction.

Published evidence supports the correlation of genome rearrangements to transformation from indolent widespread disease to more aggressive DLBCL.

Rearrangement Detection Methods

Bacterial artificial chromosome (BAC) array CGH: 2400 elements

Albertson and Pinkel, HMG 2003 12 #2 145 – 152.

Bacterial artificial chromosome (BAC) array CGH: 32,000 elements De Leeuw et al., HMG 2004 13 (17); 1827 - 37

Affymetrix 100 k SNP arrays: Genotyping and Copy number analysis.

Affymetrix 100 K Users Manual

Affymetrix 100 k SNP arrays: Genotyping and Copy number analysis.

SNP_A-1672076 (chr9 : 92630957)

SNP_A-1745782 (chr9 : 92630992)

SNP_A-1651414 (chr9 : 92691075)

SNP_A-1643223 (chr9 : 92731507)

SNP_A-1758738 (chr9 : 92840593)

SNP_A-1707255 (chr9 : 92872748)

SNP_A-1641827 (chr9 : 92872961)

SNP_A-1755966 (chr9 : 92906870)

SNP_A-1709121 (chr9 : 92926427)

SNP_A-1710928 (chr9 : 93104564)

SNP_A-1687608 (chr9 : 93104830)

SNP_A-1737371 (chr9 : 93121772)

Chr 9: 92,630,957 - 93,121,772 (490 kb)12 SNPs with average Copy Number (CN)=4.5

Min CN=4Max CN=5

Oligonucleotide approaches to detection of genome copy number imbalances (Affymetrix, ROMA, Nimblegen)

Array methods can detect genome amp-lifications and deletions. Rearrangements that do not produce a “genome imbalance” are cryptic.

Array methods do not yield reagents (clones) that can be sequenced.

>>Experiment with clone based approachesEnd Sequence Profiling (ESP)Fingerprint Profiling (FPP)

Clone fingerprinting applications

Construction of genome mapsAssessment of clone sequence assemblies

Redeye, with G. Rubin, R. Hoskins and S. Celniker

Selection and validation of tiling setsBAC array CGH (Human, Mouse….)

Cloning (for sequencing) genome rearrangements

Overview of Genome Mapping

Total Maps: 27

Total Organisms: 20

Total Fingerprints:1.8 M

Total Bases Mapped: 21 GB

Capacity: 40 human genomeequivalents pa.

genomic DNA(chromosomes)

restriction enzyme partialdigestor shearing

DNA fragments of various

sizes

size separation on agarose gels

marker

isolate DNA from appropriate gel size fraction

BAC vector

ligase

transform clones into E.

coli

array in 384-well plates (library)

•compare BAC fingerprint patterns of each clone

•identify clones containing highly related DNA and reconstruct contiguous regions of the genome (FPC)

•overnight culture•purify BAC DNA•restriction enzyme digest•agarose gel electrophoresis•restriction fragment identification

fingerprinting

map construction

Clone Fingerprinting

Fingerprint Data Generation and Restriction Fragment Identification

Fingerprinting Gel

29,950

540

M

Automated analysis of gel images is performed by BandLeader*, customized Matlab software for accurate identification and sizing of restriction fragmentsBandleader performance was assessed on fingerprints of 322 fully sequenced BAC clones (human and mouse)

•96% of real fragments were detected (sensitivity)•96% of fragments called were real (specificity)•96% accuracy in detection of co-migrating fragments, including a cluster of 11 fragments

*D. Fuhrmann et al. - Genome Research, 2003

121 lanes (96 samples + 25 marker lanes); HindIII digest; 1.2% agarose; run for 7 hours at 3.5 volts/cm in 1xTAE; gels stained post electrophoresis with SYBR Green I; images collected on MD Fluorimager 595

Throughput

Fingerprints

Human Genome

Equivalents

Day 3,000 0.15

Week 15,000 0.75

Year 800,000 42

Redeye identifies BACs with fingerprints inconsistent with their in-silico restriction maps

localized inconsistency

fingerprints consistentconfirm BAC sequence

BAC A

BAC B

sequence restriction fragments arecoloured by the relative size distanceto the nearest matching fingerprint fragment

Experimental plan

plan incorporates multiple levels of discovery, validation and comparison

compare

Lymphoma genomicDNA

Choice informed by:•arrays

•genomics•literature

•cytogenetics•relevance to progression

Array methods and ESP / fingerprinting can identify rearrangement-bearing BACs for sequencing

End Sequence Profiling

TUMOR

Volik, S. et al. 2003. End-sequence profiling: sequence-based analysis of aberrant genomes. Proc Natl Acad Sci 100: 7696-7701.

Alignment of Fingerprints to “Electronic Digests” of the reference Human Genome Sequence

experimentalfingerprint

electronicfingerprint

genome sequenceassembly

CA

TG

CA

CA

TTC

CTG

CTG

TC

ATC

CC

AA

TC

ATG

GTG

GC

CA

AC

TTTG

GA

GTC

TC

CTTG

GA

GA

GC

CTG

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AC

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TC

AG

TG

GC

TG

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AG

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AC

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AT

GC

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AG

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GTG

TA

GG

CTG

CTTC

CG

GG

TC

TC

ATC

TC

AG

ATC

CC

CG

CC

AG

TT

CTG

GC

TG

GC

GC

TG

TG

TC

AC

CTC

TTC

TC

TG

TG

TC

AG

GA

TC

ATTTTTA

TC

CC

TC

TC

TG

TC

TG

TC

TTTC

TG

TC

TTTC

CC

TG

TG

CC

CTC

CTTTC

TTC

CC

CG

GA

CC

AG

CTA

TTTC

AG

ATTC

CA

TTC

AA

CTC

TG

TTC

AG

TG

ATG

CTG

CC

GC

TC

TC

AA

TG

CG

GTTA

GA

GC

GC

AA

GA

TG

TG

AG

AA

CG

TC

TG

TG

CTG

AG

TG

GC

CTA

AA

CA

CTG

AA

GG

CTG

CG

GG

TC

TTTC

TA

ATTTC

AG

CA

TTG

AG

AC

TTTA

CA

AG

TC

CA

CA

TTC

TTG

GC

ATTG

CC

AA

CC

AG

TTA

GA

ATA

GA

AC

AA

TA

AA

TC

CC

AG

TTT

TTG

TC

ATG

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TC

TG

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ATTA

AA

ATG

GC

AA

CTG

GA

AC

AA

GG

CA

GTC

AC

T

Needleman-Wunsch

global alignment

TUMOR NORMAL

align fingerprintTUMOR

Potentially confounding issues

Repeated sequences

Sensitivity and specificity of mapping BAC fingerprints to genomeMultiple enzyme digests optimal

Clone artifactsRedundant representation of regions in independently derived clones

Repeats, Sensitivity and Specificity

Approximately 47% of genome sequence is composed of repetitive DNA sequences.

3% of repeat content is in blocks > 7.4 kb (2X the average size of a fingerprint fragment) 27% of repeat content is in blocks >1,800 bp (2X the average size of an end sequence read)

ESP is substantially affected by repeats: - 27% of end reads will have ambiguous alignments to the genome sequence- 47% of BACs will have one or neither of their end sequences unambiguously aligned, and thus will

not be useful.

Is FPP affected by repeat content? The performance of FPP alignments was assessed using a set of 43,000 simulated 130 kb BAC clone fingerprints derived from the sequence assembly

FPP alignment sensitivity - 99.8% of all BACs had associated alignments in the correct location

FPP alignment specificity- 78% of all alignments do not extend past the actual edges of the BAC - 87% of all alignments do not extend by more than 1kb (e.g. 500bp on both ends)- 99% of all alignments do not extend by more than 10kb (e.g. 5kb on both ends)

FPP: less sensitive to repeats (reduced attrition due to repeats) and samples more of clone insert

Double digest fingerprints yield specific patterns

Number of genome fragmentsmatching fragment F

bin(kb)

total size of duplicated

patterns (Mb)

10 170

15 13

20 7

30 2

small regions of the genome produce unique patterns

larger domains yield more specific patterns

EcoRI/NcoI

5-fold fingerprinting provides coverage by at least 2 BACs for 93% of the genome

simulated 50X MboI library 500 iterations

simulation shows 96% coverage at 2X+

fp depthcoverag

eall

coverage 2X+

3X 93 76

4X 97 87

5X 98 93

Clone redundancy captures rearrangements in more than one BAC

internal validation

Redundancy and sensitivity

pos A pos B

pos A pos B

pos A pos B

pos A pos B

100 kb 100 kb

50 kb 150 kb

25 kb 175 kb

10 kb 190 kb

what is the smallest mappable fragment?

At ~ 5X, 94% of all breakpoints >20 kb from edge of nearest BAC

FPP Alignment Method (v 0.1)

MCF7 Proof of concept

607 clones from MCF7 breast cancer cell line fingerprinted with 5 enzymes C. Collins and S. Volik (UCSF) all clones subjected to ESP analysis HindIII, EcoRI, BglII, NcoI, PvuII

enzymes selected to optimize sampling resolution, coverage by sizeable fragments, band spacing, robustness in laboratory

fingerprints were compared to the reference human genome sequence to map the clones onto the genome and identify BACs containing putative genome rearrangements. 206/607 BACs were identified as containing candidate rearrangements 245/607 BACs identified by ESP

148/206 were also identified by ESP as containing rearrangements.

complex rearrangements not detected by ESP were found

Complex (potential) rearrangements

M0012O05 localized to chrs 1p13.3 17q23.2 20q13.2 alignments using BglII, EcoRI, HindIII, NcoI, PvuII

1 107.21 T |210000....|219146....|225959....|237503....|246728....|252248....|260278....|270933....|281502....|288004.... 1 107.21 n ............,,,,,,,,,,,,S,,,,,,,,,,xxxxxxxXXXXXXXxxxxxXXxxXXXXXxxxXXXXXxxxxxxxxxxxxxxXxxxxxXX...,,,,,,,,,,,,,, 1 107.21 e ......,,,,,.........,,,,,,,.....,,,,,,,xxxxxxxxxxxxxxxxxxxxxXxxxxxxXXXXXSXXXSXXXXXXXxxxxx,,,,,,,,,,,,,,,,,,,,, 1 107.21 p .,,...........s...,,,,,..............XSSXXXXXXxxxxxxxssxxXXxxxxxXxxxxXXXXXXXXXXXXXXXXXXXXXXX..,,,,.........,,, 1 107.21 h ....sSS,,,,,,...,,,,,SXXXX..,S,,,,xxxxxxxxxXXXXX..sxxxxxxxxxxxxxxxxxxxxxxxx,,,xxXxxxxxxx,,.......ssSsS,,,,SS,, 1 107.21 b ..,,,,,,S,.......,,............,,...XXXXXxxxXSSXXXXXxxxxxxxxxxXXXXXXxxXXXXxxxxx,,,xXXXX........,,,,,,,,Ss...., 1 107.21 P ..................................********************************************************....................

17 59.28 T |280000....|292135....|303038....|318237....|328080....|335700....|348112 17 59.28 n .......,,,,,,,,,,,xxXXX....XXXXXxxxxXXXxx,,,,,...ss.......,,,,S,,,,,, 17 59.28 e ..s.sS,,,,....,.,,,,,,,,,,xxxxxxxxxxxxxxxxxxxxx,,,,,,...,,,......,... 17 59.28 p .........,,.............XXXXXXXxxsxxxX......,,,,,,.....,,............ 17 59.28 h .............,,,,xxxxXXXXXXXxxXXXXXxxxxxxx,,,...,,,,........,,,,,,,,S 17 59.28 b .,,,,,,,....,,,,,,,xxxXXXxxxxXXXXXXXXXXX...,,,,,,,,,,,.......,,,...,, 17 59.28 P ..................*************************..........................

20 53200000 T |200000....|205896....|215551....|223167....|233993....|240978....|244492....|250169....|259400....|262360....|268239....|27820 53200000 n ...,,,,,,,,,,.....,,,,,,,,xxxxsS,,,,,,,,,xxxxxxXXxxXxxxxsxxXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXSssxxxxxxxxxxxxxxxxXXXxxxxxxXXXXXX20 53200000 e SSXXXXXXXXXX........,,,,,,,......sss....ssxxxxxxxxxxxxxxxxxxxxxXXXXxxxxs.........................XXXXXXxxxxxxXXxxxxXXXxxxXXXX20 53200000 p xxxxsxxxxxxxxx,,S,,,,..sxxxxX.....sxxxxxxxxxxsxxxxXXXXxxxxxxXXXXxsxxxxxxxxXXXXXSSssxxxxXxxxxxxxxxxsSXXxxxXXS,,,,,,..XXXXXXXXX20 53200000 h S,,,,,,,SX.....,,,,,,,...............XXXXXX.....XXXXXxxxxxXXXXXXXXXXXxxxxXXXSXXXXXXXSsxxxxXXXxxxXXXXXxxxxxxxxxxxXXXXXxxxxxxXX20 53200000 b ......sxxxx,,,,,,,,xxxxxx,,,,,,,,,,,,,SXXXXXxxxxxxxxxxxXXXXXXS,,,,,,xxXXXXXXXXXXSSXXXXXXXxxxxxxXXXXXXXXXxxXXXXXXXXXXXXXXSSssx20 53200000 P ........................................****************************************.********************************************

..|278694....|285532....|290615....|296348....|305699....|309888....|315411....|327575....|333647....|336548....|346115. XXXXXXXXXXxxxXXXXXXXXXXxxxxXXXXXXXXXXXXxxxxxxsss...sSXXXXXxxxxXXXXXXXXX....,,,,,,,.......XXXXXXXXXXXXX.................. xxXXXXxxxxxxxxxxXXXxxxxxxxxxsxxxxxxXXXxxxxxxxxxxx,,,,,,,,xxsxxx,,,,,,,,,,,..,,SsS,,,,,,,...ss....,,,,,,,,,,........sS,,S XXXXXXXSXXXXXXSssxxxXXxxx,xxxxxxXXxxxxxxxxxxXXxxxx,,,,SSXXXXXXXXSSSXXxxxx,,,,......sSS,,,,.............XX...s.,..,....,, xxxxXXXXXXXSXXXXXXXXXXXXxxxxxxxxxXXXSXXXxxXXXXXxxxxxxxxxxxxxxXXXXSXXxxxx,,,,,,,,,,,,,.........,,,,Ss......,,,,,,,,,,,,,, XSSssxxxxXXXXXXXXXxxxXXXXXXXXXSXXXXXXXXXXxxXXXXXXXXXXXXxxxxxxxxxxxxxxx,,,,,,,,,,,,,,,,,.....,,,S,,,,,...,,,,,,,,........ ***********************************************************************.................................................

Visual comparison of results obtained from fingerprint and ESP methods.

Both methods are capable of detecting rearrangements not found by the other method.

Not all MCF-7 clones harboured rearrangements. Clones in pilot project were enriched for those with specific rearrangements on chrs 1, 3, 17 and 20.

Rearrangement profile of MCF-7 genome is known to be extremely complex.Davidson, J.M. et al. 2000. Molecular cytogenetic analysis of breast cancer cell lines. Br J Cancer 83: 1309-1317.

Progress: FPP analysis of FL

fingerprinted clones(EcoRI, NcoI)

50,30050% of target

clones with average fp size 60-260kb

46,501 (92%)

clones 60-260kb with an alignment >25kb

45,509 (98%)~2.1X

unique coverage 2.2 Gb (77%)

clones with multiple alignments (both >25kb)

intra-chromosomal 189 (0.4%)

inter-chromosomal 967 (2.1%)

Library #1:patient with cytogenetic profile showing only t(14;18); est average insert size 135 kb

Library #2: patient showing complex cytogenetic profile in addition to t(14;18); est average insert size 130 kb

90% of clones are in the range 75-225 kb

Empty wells < 1%

Failures:3.6 % vs. 7% (All)

FPP alignments currently cover 77% of the genome

average

Redundant coverage

Coverage bp >=1 2,216,990,483>=2 1,344,663,365 >=3 654,423,007>=4 264,307,507>=5 90,817,363>=6 27,173,488

candidate FPP rearrangements

inter-chromosomaln=967

intra-chromosomaln=189

Clone size distribution: AEX HT0001

Clones associated with candidate rearrangements tend to be long

Proximity of BAC alignments to segmental duplications

Shorter clones are more likely to be associated with a segmenatl duplication

Redundancy of sampling resolves inconsistent alignments due to chimeric clones

T0049G09 aligns to chrs 5 and 14 alignment on chr14 is embedded within a contig formed by clones with single alignments breakpoint suggested by T0049G09 is inconsistent with neighbouring alignments

Translocation seen in FL t(14;18)(q32.33;q21.33)

BCL-2

IGH

FPP alignments of clone T0099C19

chr 18

cloneregion T0099C19 18 58898198-58941792 size 43595 score 113.6433

18 58880000 T |880000....|906215....|921342....|932071....|951463. 18 58880000 e X,..,S,XXXxXXXXxxXXXxx..xxSsSxxSxXXXsX,,,,.,,.....,. EcoRI18 58880000 n ...,,,,,XxxxXxXXxxS,,..xxXXXXXxxxxSxxxxSx...,,.xS,,, NcoI18 58880000 F 1000000147************8****************9*61000010000 18 58880000 F 1000038***************8************85410100000010000 18 58880000 P .......**********************************...........

chr 14

cloneregion T0099C19 14 105464595-105493586 size 28992 score 96.0589 14 105454680-105464594 size 9913

14 105440000 T |440000....|451148....|467323....|476363....|491061 14 105440000 e ....,,,,,,,,,,,,,,,SxxxxxxSxxXXsSxxXXsSxxxxxx..,,. EcoRI14 105440000 n .sS,,.s.,.,.xSxXx.xxxSsX,XXXxxXXXXxxXXXXxSsSxx..,, NcoI14 105440000 F 00000000000010134013679***********************8400 14 105440000 F 00000000000497**97***********************876410000 14 105440000 P ............**********************************....

chr 18

3’ BCL-2BES @ 58,892,7715.4 kb away

chr 14

IgH BES @ 105,494,7031.1 kb away

Double digests:

EcoRI+NcoI double digest, 0.9% Trevi gel, 5.3 V/cm, 4 hour

sensitivity: 91.1% / 98.1% (c.f. 96.5% single)specificity: 88.2% / 93.8% (c.f. 96.2% single)

12 kb

0.43 kb

>0.43 kb >1.00 kb

Conclusions

A BAC fingerprinting – based approach can identify rearranged clones (MCF7 and lymphoma).

BAC libraries can be made from primary tumor material (2 lymphoma libraries).

Such libraries can be subjected to high throughput BAC fingerprinting.

The fingerprints can be scanned for BACs bearing candidate genome rearrangements.

FISH has confirmed ~50% of candidate rearrangements.

Redundancy is an asset!

Acknowledgments

BC Cancer Agency Joseph Connors Randy Gascoyne Doug Horsman

UCSF Comprehensive Cancer Centre Colin Collins Stas Volik

Joe Gray

BC Cancer FoundationMichael Smith Foundation for Health Research

National Human Genome Research Institute (USA)Genome Canada / Genome BC

BCCA Genome Sciences Centre

Genome Mapping Group Martin Krzywinski Jacquie Schein

DNA Sequencing Group Rob Holt George Yang