Figure 1
Next-generation DNA sequencing workflow. Genomic DNA samples are input to a molecular inversion probe capture reaction. Each target (depicted by blue and orange regions) is captured by multiple probes that anneal to nonoverlapping genomic intervals. PCR is performed using primers containing patient-specific barcodes, yielding barcode libraries (turquoise and purple). Equal volumes of the libraries are pooled and enter the Illumina HiSeq high-throughput sequencing workflow. Following sequencing, reads enter either the alignment only (AO, left) or Genotyping by Assembly-Templated Alignment (GATA, right) analysis pipeline. AO first partitions reads by sample molecular barcode, then in parallel for all samples performs short-read alignment, base-quality recalibration, realignment around putative indels, and genotyping. GATA partitions reads first by sample molecular barcode, then by target. Reads are assembled into contiguous sequences (contigs) that are then aligned to the reference genome. Raw reads are then aligned to the contigs, and raw-read mapping and variant information relative to the reference are determined using reference-contig and read-contig alignments. Finally, base-quality score recalibration and genotyping are performed on the mapped, raw reads. gDNA, genomic DNA.
