Open Access Highly Accessed Methodology article

Massively-parallel sequencing of genes on a single chromosome: a comparison of solution hybrid selection and flow sorting

Jamie K Teer13*, Jennifer J Johnston1, Sarah L Anzick2, Marbin Pineda2, Gary Stone2, NISC Comparative Sequencing Program11, Paul S Meltzer2, James C Mullikin1 and Leslie G Biesecker1

Author affiliations

1 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

2 National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

3 Current address: H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

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Citation and License

BMC Genomics 2013, 14:253  doi:10.1186/1471-2164-14-253

Published: 15 April 2013

Abstract

Background

Targeted capture, combined with massively-parallel sequencing, is a powerful technique that allows investigation of specific portions of the genome for less cost than whole genome sequencing. Several methods have been developed, and improvements have resulted in commercial products targeting the human or mouse exonic regions (the exome). In some cases it is desirable to custom-target other regions of the genome, either to reduce the amount of sequence that is targeted or to capture regions that are not targeted by commercial kits. It is important to understand the advantages, limitations, and complexity of a given capture method before embarking on a targeted sequencing experiment.

Results

We compared two custom targeted capture methods suitable for single chromosome analysis: Solution Hybrid Selection (SHS) and Flow Sorting (FS) of single chromosomes. Both methods can capture targeted material and result in high percentages of genotype identifications across these regions: 59-92% for SHS and 70-79% for FS. FS is amenable to current structural variation detection methods, and variants were detected. Structural variation was also assessed for SHS samples with paired end sequencing, resulting in variant identification.

Conclusions

While both methods can effectively target genomic regions for genotype determination, several considerations make each method appropriate in different circumstances. SHS is well suited for experiments targeting smaller regions in a larger number of samples. FS is well suited when regions of interest cover large regions of a single chromosome. Although whole genome sequencing is becoming less expensive, the sequencing, data storage, and analysis costs make targeted sequencing using SHS or FS a compelling option.

Keywords:
Flow sorting; Flow cytometry; Targeted-sequencing; Sequencing; Genomic-capture; Chromosome; Genome