Breakpoint analysis of balanced chromosome rearrangements by next-generation paired-end sequencing.
Characterisation of breakpoints in disease-associated balanced chromosome rearrangements (DBCRs), which disrupt or inactivate specific genes, has facilitated the molecular elucidation of a wide variety of genetic disorders. However, conventional methods for mapping chromosome breakpoints, such as in situ hybridisation with fluorescent dye-labelled bacterial artificial chromosome clones (BAC-FISH), are laborious, time consuming and often with insufficient resolution to unequivocally identify the disrupted gene. By combining DNA array hybridisation with chromosome sorting, the efficiency of breakpoint mapping has dramatically improved. However, this can only be applied when the physical properties of the derivative chromosomes allow them to be flow sorted. To characterise the breakpoints in all types of balanced chromosome rearrangements more efficiently and more accurately, we performed massively parallel sequencing using Illumina 1G analyser and ABI SOLiD systems to generate short sequencing reads from both ends of DNA fragments. We applied this method to four different DBCRs, including two reciprocal translocations and two inversions. By identifying read pairs spanning the breakpoints, we were able to map the breakpoints to a region of a few hundred base pairs that could be confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. Our results show the feasibility of paired-end sequencing of systematic breakpoint mapping and gene finding in patients with disease-associated chromosome rearrangements.
Chen
Wei
W
Max Planck Institute for Molecular Genetics, Berlin, Germany. wei.chen@mdc-berlin.de
Ullmann
Reinhard
R
Langnick
Claudia
C
Menzel
Corinna
C
Wotschofsky
Zofia
Z
Hu
Hao
H
Döring
Andreas
A
Hu
Yuhui
Y
Kang
Hui
H
Tzschach
Andreas
A
Hoeltzenbein
Maria
M
Neitzel
Heidemarie
H
Markus
Susanne
S
Wiedersberg
Eberhard
E
Kistner
Gerd
G
van Ravenswaaij-Arts
Conny M A
CM
Kleefstra
Tjitske
T
Kalscheuer
Vera M
VM
Ropers
Hans-Hilger
HH
D001483:Base Sequence; D002675:Child, Preschool; D002869:Chromosome Aberrations; D019457:Chromosome Breakage; D005260:Female; D015321:Gene Rearrangement / Q000235:genetics*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008969:Molecular Sequence Data; D011247:Pregnancy; D017422:Sequence Analysis, DNA / Q000379:methods*