Fig. 1
The primer-barcode assembly method for targeted amplification by PCR. a The principle of combinatorial indexing of a set of amplicons (Gene1-GeneX) using panels of forward (m × BcF) and reverse (n × BcR) DNA barcodes, which are used to tag invariant forward and reverse multiplexed primer sets, respectively. The predetermined targets are amplified by multiplex PCR (inset), pooled, and analyzed by NGS (any platform). b Primer-barcode assembly in detail: a barcode and a set of reverse complementary (rc) primers (only one is shown) are hybridized via 10-mer adapter, followed by fill-in DNA synthesis of the two strands by the Klenow fragment (an A base is frequently added to the 3′ ends). rc strands with 5′P ends are preferred substrates of λ-exo, which thereby produces barcoded single-stranded gene-specific primers. *Optional trinucleotide “protection group” that inhibits λ-exo digestion (Additional file 7: Figure S1d). c Gel electrophoresis demonstrating the intermediate products of the assembly process: rc primers (P) and barcodes (B) following hybridization (P + B), Klenow fill-in (K, increasing the molecular weight), and heat inactivation (KHI). λ-exo treatment, which creates single-stranded barcoded primers (λ, reduces the molecular weight), and heat inactivation (λHI). Samples are a single barcode linked to an adapter and a single rc primer linked to an rc adapter, ran on 2.5% agarose gel with GeneRuler™ 100 bp DNA Ladder (L). d Co-amplification of 10 loci in BRCA1 and BRCA2 from gDNA using primers assembled with combinations of two forward (L03 and L08) and two reverse (R01 and R06) barcodes, and assessment of the products by qPCR using nested primers. Non-pre-amplified gDNA, non-barcoded rc primers, and non-targeted loci (MSX1 and ZIC1) are negative controls. e Assessment of the efficiency of primer synthesis as a function of the number of multiplexed primers. Primer set size was tested for the range of 1 to 10 (increments of 1), starting with Amp3 as singleplex, with the order shown in the right pane. The concentration of the individual primers was equal in all reactions, and the barcode concentration was matched to the total primer concentration. Non-pre-amplified gDNA and the non-targeted DNMT3B locus were used as negative controls. Error bars represent the standard deviation of three replicates