Additional file 1 of LUMI-PCR: an Illumina platform ligation-mediated PCR protocol for integration site cloning, provides molecular quantitation of integration sites

Additional file 1: Figure S1. Step by step summary of LUMI-PCR library construction including all adapter and primer sequences. a) The figure depicts an LTR (blue bases) containing DNA fragment with a variable number of bases of genomic DNA (black X(X)nX bases) and a 5′ LTR sequence (blue). DNA is sheared by sonication. Overhanging ends are blunted, cleaned and then A-tailed (yellow highlighted bases) and cleaned. b) Adaptors containing unique indices (light green XXXXXXXXXX bases) and UMIs (dark green NNNNNNNN bases) are ligated to A-tailed DNA using T overhangs. c) Ligated fragments containing MuLV LTR sequence are bound by the LTR primary PCR primer. The first strand is synthesized from the LTR bound primer creating a lower strand that is compatible to the adapter primer. d) After the first strand of synthesis, the adapter primer and LTR primer can then amplify with exponential kinetics. Non-LTR containing fragments in the ligation are not amplified. e) A nested secondary PCR primer is used to add a second index sequence (light green XXXXXXXXXX bases) to the primary PCR product and further amplify the sequences. d) The secondary PCR products are quantitated, libraries are pooled and then loaded onto a MiSeq or HiSeq flow cell and clusters are generated. e) After cluster generation the first strand acts as the template for read 1 (the adapter end sequencing primer), index 1 (sequencing the LTR index added during the secondary PCR) and index 2 (the adapter index including the UMI sequenced from the flow cell primer). The index 2 read is a non-standard 18–20 bp, instead of the usual 10 bp, to include the UMI sequence. f) After strand regeneration, read 2 sequences the LTR-genome junction (using an LTR primer that can be placed at the junction or offset back from the junction). Figure S2. Graphical summary of informatics pipeline used to process reads into integration sites. Detailed step by step instructions for executing all scripts are available at https://github.com/anthonyuren/LUMI-PCR-pipeline/. Figure S3. Plate layouts for Beckman Biomek liquid handling workstation (below) Plate layouts for each program are listed on the next 4 pages. All programs begin with a box of tips loaded in the tip loader. Some programs require replacement of the tip box 30 min into the protocol. Detailed step by step protocols can be obtained by loading the .xpl files for each protocol into the Beckman Biomek software. Table S1. Diverse studies employ ligation-mediated PCR protocols for positioning of mobile genetic elements, viruses, transposons and transgene vectors. Table S2. Summary statistics for each library. Replicate libraries were prepared from a single spleen DNA sample from an MuLV-infected mouse. The number of reads, unique DNA fragments and integration sites are summarized.