Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing

Abstract

Massively parallel DNA sequencing technologies have greatly increased our ability to generate large amounts of sequencing data at a rapid pace. Several methods have been developed to enrich for genomic regions of interest for targeted sequencing. We have compared three of these methods: Molecular Inversion Probes (MIP), Solution Hybrid Selection (SHS), and Microarray-based Genomic Selection (MGS). Using HapMap DNA samples, we compared each of these methods with respect to their ability to capture an identical set of exons and evolutionarily conserved regions associated with 528 genes (2.61 Mb). For sequence analysis, we developed and used a novel Bayesian genotype-assigning algorithm, Most Probable Genotype (MPG). All three capture methods were effective, but sensitivities (percentage of targeted bases associated with high-quality genotypes) varied for an equivalent amount of pass-filtered sequence: for example, 70% (MIP), 84% (SHS), and 91% (MGS) for 400 Mb. In contrast, all methods yielded similar accuracies of >99.84% when compared to Infinium 1M SNP BeadChip-derived genotypes and >99.998% when compared to 30-fold coverage whole-genome shotgun sequencing data. We also observed a low false-positive rate with all three methods; of the heterozygous positions identified by each of the capture methods, >99.57% agreed with 1M SNP BeadChip, and >98.840% agreed with the whole-genome shotgun data. In addition, we successfully piloted the genomic enrichment of a set of 12 pooled samples via the MGS method using molecular bar codes. We find that these three genomic enrichment methods are highly accurate and practical, with sensitivities comparable to that of 30-fold coverage whole-genome shotgun data.

Exome sequencing and SNP analysis detect novel compound heterozygosity in fatty acid hydroxylase-associated neurodegeneration

“…12,13 This information can be used in combination with other genomic evaluations to identify candidate genes for complex cases. We report a case where ES and SNP array analyses were used together along with phenotypic information to make a diagnosis of an extremely rare disorder.…”

“…Sequence alignment, genotype calling, and annotation were performed at NISC and are described elsewhere. 12,13 Additional annotation and prioritization of genotypes according to disease-causing potential was performed using methods described earlier 13 and with VAR-MD (Sincan M et al, unpublished data). The dbSNP132 database (that includes data from the 1000 genomes project) and a local database of 177 exomes were used to exclude common DNA variants.…”

Individualized Iterative Phenotyping for Genome-wide Analysis of Loss-of-Function Mutations

“…Solution hybridization exome capture was performed with either the SureSelect All Exon System (Agilent Technologies) or the Illumina TruSeq system (Illumina). Flow cell preparation and paired-end read sequencing were performed with either the GAIIx or HiSeq 2000 sequencer 7 (Illumina). Image analyses and base calling were performed as described.…”

“…Image analyses and base calling were performed as described. 7 Reads were aligned to hg19, NCBI 37, via novoalign (Novocraft Technologies). Samples were sequenced to sufficient coverage such that 85% of the targeted exome was called with high-quality variant detection (reported as genotype at every callable position).…”

P2‐030: Investigating the Role of Clu, Picalm, and Cr1 in Alzheimer's Disease

“…Practically, SS is faster, requires less in the way of specialist equipment and has lower DNA requirements than its array based counterpart (Mamanova et al 2010;Teer et al 2010). In terms of performance, SS's recurrent downfall is that, due to the stringent repeat masker (discussed in greater detail in section 2.4) used in the generation of target regions for bait design, often less of the region of interest is targeted by this method when compared to NG, which uses its own, seemingly less conservative, repeat masking software.…”