Fig. 1

Comparison of LUMI-PCR with regular Illumina dual index library prep and with regular splinkerette PCR library prep. a) The steps of a traditional ligation-mediated PCR strategy using adapters with non-complementary segments and two rounds of nested PCR (e.g. splinkerette). The adapter strands are partially non-complementary and the lower strand (dark green) has no complementary primer. The adapter primer (blue) cannot bind to a template until the first strand has been synthesised from the virus primer (red). Subsequent steps will amplify virus flanked genomic regions but not other regions. b) Standard Illumina library preparation protocols for single index libraries. Using ligation of adapters, an index (black) is included in the adapter for each library, with one copy per fragment being present in the final product. Both strands are amplified yielding different termini at each end for flow cell binding (blue & purple). c) Illumina Nextera library prep using tagmentation. Adapters are added via Tn5 transposase. Both strands are amplified simultaneously using primer pairs that add an index at each end. d) LUMI-PCR is a hybrid protocol for ligation-mediated PCR that uses one index in the adapter and one in the secondary PCR step. A unique molecular identifier (UMI orange) is included adjacent to the adapter index (black) for quantitation of library fragments. The placement of the index is switched from the strand normally used in Illumina adapters such that it will be retained after the first strand synthesis from the virus primer. The flow cell binding sequence normally present in the Illumina adapter (purple) is included in the LTR primer of the secondary PCR amplification. e) A modified dual index Nextera sequencing protocol is used with custom primers and modified numbers of bases read from each index depending on the length of the custom index and the UMI (our protocol uses 10 bp indexes and an 8–10 bp UMI). The custom virus primer can be nested back from the virus genome junction to allow the junction to be sequenced