A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay

Abstract

Targeted amplicon sequencing to identify pathogens, resistance-conferring mutations, and strain types is an important tool in diagnosing and treating infections. However, due to the short read limitations of Illumina sequencing, many applications require the splitting of limited clinical samples between two reactions. Here, we outline hairpin Illumina single-tube sequencing PCR (hissPCR) which allows for the generation of overlapping amplicons containing Illumina indexes and adapters in a single tube, effectively extending the Illumina read length while maintaining reagent and sample input requirements.

Fig 1. Hairpin Illumina single-tube sequencing PCR (hissPCR).

In step 1, primers containing a universal tail sequence amplify a target up to 1200 nucleotides flanking each gene of interest. In step 2, tailed gene-specific primers target the amplicon generated in step 1, and, together with the universal tailed primers that contain the index and adapter sequence, generate two overlapping amplicons of sequenceable length while the gene-specific primers (Forward-1 [F1] and Reverse-2 [R2], each containing the same tailed sequence), form a hairpin during amplification, thus nullifying the formation of the 100 base pair overlap amplicon and allowing amplification of the intended ~600 base pair amplicons.

Fig 2. Expected results.

A) hissPCR generated amplicons using two forward and two reverse overlapping primers in a single tube. Amplicons contain either no Illumina adapter, one Illumina adapter, or two Illumina adapters per amplicon. B) The relative proportion of amplicons in the sample containing both Illumina adapters. C) Melt curves showing the expected amplicons for each primer set and the ratio of amplicons containing both Illumina adapters.