Fig. 1

Schematic representation of the methods used for NGS library construction. A cDNA library labeled with Primer IDs (top) is divided and used for each method. a uSGS. Short PCR primers (25 and 31 bases) containing 5′ dU in place of dT residues (dots in the primers) are used to amplify the cDNA. Products are cleaved at the dU sites creating dsDNA with 17-nt 3′-overhangs at both ends. The ends are then ligated to the essential NGS adapters and filled out using Klenow Fragment DNA polymerase to generate a fully double-stranded NGS library. b Long primer PCR-1. Long primers (90 and 93nt) containing NGS adapter sequences are used to amplify the cDNA library. c Long primer PCR-2. Relatively long primers (50–61nt) are used in 2 rounds of flanking PCR to amplify the cDNA and attach the adaptors. d Long primer PCR-3 involves 3 rounds of PCR. The cDNA is amplified with short primers (25 and 31 bases) followed by 2 rounds of flanking PCR using long primers (50–61nt) to attach the adaptors