Benefits and Challenges with Applying Unique Molecular Identifiers in Next Generation Sequencing to Detect Low Frequency Mutations

Abstract

Indexing individual template molecules with a unique identifier (UID) before PCR and deep sequencing is promising for detecting low frequency mutations, as true mutations could be distinguished from PCR errors or sequencing errors based on consensus among reads sharing same index. In an effort to develop a robust assay to detect from urine low-abundant bladder cancer cells carrying well-documented mutations, we have tested the idea first on a set of mock templates, with wild type and known mutants mixed at defined ratios. We have measured the combined error rate for PCR and Illumina sequencing at each nucleotide position of three exons, and demonstrated the power of a UID in distinguishing and correcting errors. In addition, we have demonstrated that PCR sampling bias, rather than PCR errors, challenges the UID-deep sequencing method in faithfully detecting low frequency mutation.

Fig 1. UID-targeted DNA sequencing workflow and the principle in distinguishing errors from true mutation.

(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.

Fig 2. Flowchart for error vs mutation data analysis.

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.

Fig 3. Error rate at each nucleotide position of FGFR3-Exon14 and Exon 7.

(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.

Fig 4. Q5 DNA polymerase improved the fidelity of UID deep sequencing.

(A) Comparison of error rate at each position of FGFR3-Exon 9 in Stage 2 PCR and Illumina sequencing when Q5 (with Taq spike in) vs Platinum DNA Polymerase was used. (B) Comparison of error rate at each position of FGFR3-Exon 9 in Stage 1 PCR when Q5 (with Taq spike in) vs Platinum DNA Polymerase was used.

Fig 5. Error rates of deletion types in FGFR3 Exon14.

Platinum DNA polymerase was used. Notice the scale is 100 fold lower than that of Fig 3 or Fig 4.

Fig 6. PCR Bias for wild type over mutant FGFR3-Exon7.

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.