Overview of widely used MPS methods. In all cases, sequencing begins with a universal sequencing adaptor ligated to the DNA to be sequenced, which allows a sequencing primer to bind. (a) 454 sequencing relies on sequential cycles of single nucleotide addition followed by wash steps. When a nucleotide is incorporated into the growing DNA strand, an ATP is released, which reacts with luciferin to release fluorescence which is captured by the imager. (b) Cyclic reversible termination, employed by the Illumina sequencing systems relies on fluorescent reversible chain terminating nucleotides. All four nucleotides are added and when a nucleotide is incorporated into the strand by DNA polymerase, nucleotide addition is halted and fluorescence imaging occurs. The fluorescence is cleaved and nucleotides are added again. (c) SOLiD sequencing uses a ligation reaction between fluorescent two-base oligonucleotides. Ligation is followed by fluorescence imaging, then a cleavage of the fluorescent moiety, and then addition of the fluorescent oligonucleotides. To ensure that all bases are sequenced by ligation, a sequencing primer shifted one position (n+1) is added and the process is repeated.

Genomic technologies used for genetic testing for deafness. (a) Single nucleotide extension microarrays use thousands of sequencing primers that are adhered to the array. DNA to be sequenced hybridizes to the sequencing primers with the single base to be sequenced overhanging. Fluorescent nucleotides and polymerase are added, allowing a nucleotide to be incorporated and imaging to occur. (b) Resequencing arrays are composed of four oligonucleotide probes for each base to be sequenced. DNA is biotin labeled and hybridized to one of the four oligonucleotide probes. The location of the biotin can then be imaged. (c) Solution-phase targeted genomic enrichment starts with genomic DNA that has been sheared and universal adaptor-ligated. In this figure, genomic regions of interest are shown in red and purple. This DNA library is combined with biotin-labelled RNA baits that have been designed to be complementary to the regions of interest. Hybridization occurs in solution and sequence capture with streptavidin-coated beads pulls the biotin-labelled baits, which are hybridized to the genomic regions of interest, out of solution. This leaves an enriched library which can then be sequenced using MPS (Figure 1).

Overview of MPS bioinformatics analysis. (a) An MPS sequencer generates millions of short sequencing reads. (b) The raw sequencing reads contain information on quality of each read. (c) Output is filtered and mapped to the reference human genome. Exon 2 of the GJB2 (connexin 26 gene) is shown here with several thousand forward (blue) and reverse (red) sequencing reads shown aligned. (d) Any bases found to be variant from the reference genome are identified and annotated. In this case, the reference base is “A”, but about 50% of reads contain a “C”, indicating an A>C change which causes the pathogenic Val167Met amino acid change.