Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection

Abstract

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.

Figure 1.

Experimental Design. Genomic DNA from nine HapMap samples was chosen for the study (three trio families). DNA from one of the samples (NA11881) was prepared twice (with and without an indexed adapter), target enriched and sequenced separately as single samples (non-indexed sample and one indexed sample in Step 1 and enriched libraries 1 and 2 in Step 2). One trio family (NA11881, NA11882 and NA10859; all indexed) was pooled after the Illumina genomic DNA sample prep and enriched together using one SureSelect enrichment reaction to produce the enriched library 3 sample. Indexed DNA from all nine samples was also pooled after the Illumina genomic DNA sample prep and enriched together using one SureSelect enrichment reaction to produce the enriched library 4 sample. Note: enriched libraries 3 and 4 were also sequenced using 80 bp reads to generate additional data for validation of the method for SNP detection.

Figure 2.

Sequence coverage across on-target and off-target regions. Sequence coverage is plotted for the single non-indexed and indexed samples at an on-target site (PTBP2 on chromosome 1; A) and at an off-target site (chromosome 12; B). Inclusion of the index does not dramatically change the pattern of sequence coverage at on-target or off-target regions. The higher sequence coverage observed for the non-indexed sample compared with the indexed sample reflects the larger number of clusters that passed QC filtering during the sequence run (Supplementary Table SB).

Figure 3.

Percentage of sequence reads per indexed sample in sequenced libraries. Percentage distribution per sample of sequence reads (pre-alignment to reference genome; 40 and 80 bp data combined) for the three-index (A) and the nine-index (B) sample libraries. The relative underperformance of sample NA10859 in the three-index library is not observed in the nine-index library and is unlikely to be due to a systemtaic problem with the ACACAT index.