7Mix_E7 (4)

4th part of 7Mix_E7 fastq dat

7Mix E7 (2)

2nd part of 7Mix_E7 fastq dat

7Mix_E7 (1)

1st part of 7Mix_E7 fastq dat

7Mix_E7 (3)

3rd part of the 7Mix_E7 fastq dat

UID-targeted DNA sequencing workflow and the principle in distinguishing errors from true mutation.

<p>(A) Illustration of UID-targeted DNA sequencing workflow. (B) True mutation from errors introduced during PCR and sequencing. A true mutation (illustrated as red star) is expected to be present in all the reads carrying the same UID (or derived from the same template molecule), while an error (illustrated as blue star) is expected in some but not all the reads carrying the same UID.</p

Flowchart for error vs mutation data analysis.

<p>Reads were grouped by UID. When an UID has 3 or more reads, the ratio of altered reads/total reads was calculated. If the ratio was more than 95%, the altered nucleotides were counted as pre-existed in the template tagged with the UID; if the ratio was less than 95%, the altered nucleotides were counted as error occurred during the amplification of the tagged template or the sequencing step.</p

PCR Bias for wild type over mutant FGFR3-Exon7.

<p>FGFR3-Exon7 was amplified from the wild type template, mutant template (Chr4:1803564 G>A), or 1:1 mixture of the wild type and mutant templates, and the PCR products were sequenced by Sanger method.</p

Error rate at each nucleotide position of <i>FGFR3</i>-Exon14 and <i>Exon 7</i>.

<p>(A) Error plotted for all 114 nucleotides of Exon14 (amplified with Platinum Taq), with 30 nucleotides magnified. (B) Error plotted for all 112 nucleotides of Exon7 (amplified with Q5 enzyme), with 30 nucleotides magnified.</p