Using Primer ID Approach to Reduce PCR/Sequencing Errors and Skewing and Detecting Sampling Depth
cDNA was synthesized using primers with a block of degenerate nucleotides (Primer ID), followed by two rounds of PCR amplification to incorporate MiSeq adaptors and 6 nucleotide-long indexes for multiplexing of different samples for the same sequencing run. In addition, we added a 4-nucleotide degenerate block in the forward PCR primer. Paired-end sequencing started from the degeneracies present at both ends, allowing individual amplicons to be detected as distinct sequencing clusters.  RSB14, RSB16, RSB18, RSB20, RSB22,RSB24 are 6 runs using 8E5 cell supernatants as template to measure Primer ID approach residue error rate at HIV-1 env gene V1 through V3 region. Three sets of reverse transcriptase enzyme and PCR primers were used.  Set1, Superscript III plus KAPA2G robust. RSB14 and RSB16 are two repeats. Set2, Supperscript III plus Phusion polymerase. RSB18 and RSB20 are two repeats. Set3, AccuScript plus KAPA2G robust. RSB22 and RSB24 are two repeats. RSPRCT1 and RSPRCT3 are two runs using HIV-1 virus extracted from 8E5 cell supernatants as template to measure residual error at HIV-1 pol gene protease coding domain.  RSRTV02 and RSRTV03 are sequencing data from one protease naive patient plasma sample (two separate cDNA reactions). RSD01 to RSD11 are sequencing data from one plasma samples with serial dilutions. RSD01, RSD02, RSD03 are sequencing data with 1:1 dilution of original plasma with 10,000 copies of virus for each reaction. RSD04, RSD05, RSD06 are 1:3 dilution, which is 3,333 copies of virus as initial template. RSD07, RSD08, RSD09 are 1:9 dilution, which is 1,111 copies of virus as initial template. RSD10 and RSD11 are 1:27 dilution, which is 370 copies of virus as initial template.  RSPRCT5 to RSPRCT12 are 8 repeats of sequencing of viral RNA extracted from 8E5 cell supernatants at HIV-1 protease coding domain. The purpose of these runs is to study the utilization of Primer ID and to investigate any possible sequence patterns of Primer ID which may be favorably amplified by PCR.