To improve accuracy
PGT’s mutation verification tagging technology relies on the labelling of each individual nucleic acid molecule (template) in the starting sample with a uniquely identifiable DNA tag which is maintained during subsequent manipulations, including polymerase chain reaction (PCR) and next generation sequencing (NGS). Following NGS, sequencing read data is first computationally grouped into shared unique-tag sequence ‘read-groups’. As the reads within a read-group are derived from a single starting nucleic acid molecule, they necessarily should have the same DNA sequence. If a variant is present in one or a few of the reads of a read group, it is highly likely to be an anomaly created by an error introduced in sample preparation or sequencing and the observed change is discarded. Thus this technology allows for the detection and high confidence verification of genuine rare variants. Analysis of ungrouped NGS reads does not provide information on the diversity or number of starting templates measured. Assembly of NGS reads into read-groups using unique template-tag establishes a 1:1 relationship with starting templates, and as such provides quantitative data on the number and diversity of templates in the starting sample.
PGT’s mutation verification tagging technology features include:·
- High confidence (high sensitivity, high specificity) rare variant determination. Template tagging has been used to identify bona fide mutations that are 100x less abundant than the ~3% frequency which can be determined by standard NGS at high specificity. In infectious disease treatment, knowledge of mutations below 1% in an infection can be clinically significant in predicting drug failure and can inform therapeutic decision-making.·
- Template normalization. Mutation verification template tagging allows the determination of the actual number of unique starting templates sequenced, which cannot be determined from standard NGS. Knowing this allows ascertainment of variant calling confidence and assay performance, i.e. that sufficient unique molecules were sequenced to make a valid variant call at any given frequency. This is particularly important when dealing with starting materials that have low numbers of template, such as a low titer viral infection or dilute samples, and provides a valuable ‘internal quality assessment’ for clinical assays. It is also very much an issue when many samples are multiplexed together.·
- Counting of starting templates. Knowing the true number of unique starting molecules provides a count of initial templates and in assays configured to detect multiple infective agents or strains, allows accurate comparison of the relative abundance and population structure of different infective agents in a mixed population.
- Casbon et al., (2011) “A method for counting PCR template molecules with application to next-generation sequencing.” Nucleic Acids Research 39(a12):e81
Examples of Issued US Intellectual Property
- “Increasing confidence of allele calling with molecular counting” US issued patents 8,481,292 and 8,685,678
- “Method for accurately counting starting molecules” US issued patent 8,715,967
- “Method for preparing a counter-tagged population of nucleic acid molecules” US issued patent 8,722,368
- “Method for adding DBRs by primer extension” US issued patent 8,728,766
- “Method for tagging using split DBRs” US issued patent 8,741,606
- “Methods and compositions for tagging and identifying polynucleotides” US issued patent 8,476,018
- “Molecular counting” US issued patent 7,537,897