Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays.
We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes.
Bau
Stephan
S
febit biomed gmbh, Im Neuenheimer Feld 519, 69120, Heidelberg, Germany.
Schracke
Nadine
N
Kränzle
Marcel
M
Wu
Haiguo
H
Stähler
Peer F
PF
Hoheisel
Jörg D
JD
Beier
Markus
M
Summerer
Daniel
D
D001483:Base Sequence; D053938:DNA Fragmentation; D018390:Gene Targeting; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D016158:Genes, p53 / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D044085:Microfluidics / Q000379:methods*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*