June 1, 2021  |  

A simple segue from Sanger to high-throughput SMRT Sequencing with a M13 barcoding system

High-throughput NGS methods are increasingly utilized in the clinical genomics market. However, short-read sequencing data continues to remain challenged by mapping inaccuracies in low complexity regions or regions of high homology and may not provide adequate coverage within GC-rich regions of the genome. Thus, the use of Sanger sequencing remains popular in many clinical sequencing labs as the gold standard approach for orthogonal validation of variants and to interrogate missed regions poorly covered by second-generation sequencing. The use of Sanger sequencing can be less than ideal, as it can be costly for high volume assays and projects. Additionally, Sanger sequencing generates read lengths shorter than the region of interest, which limits its ability to accurately phase allelic variants. High-throughput SMRT Sequencing overcomes the challenges of both the first- and second-generation sequencing methods. PacBio’s long read capability allows sequencing of full-length amplicons

June 1, 2021  |  

TLA & long-read sequencing: Efficient targeted sequencing and phasing of the CFTR gene

Background: The sequencing and haplotype phasing of entire gene sequences improves the understanding of the genetic basis of disease and drug response. One example is cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have revolutionized CF treatment, but only in a minority of CF subjects. Observed heterogeneity in CFTR modulator efficacy is related to the range of CFTR mutations; revertant mutations can modify the response to CFTR modulators, and other intronic variations in the ~200 kb CFTR gene have been linked to disease severity. Heterogeneity in the CFTR gene may also be linked to differential responses to CFTR modulators. The Targeted Locus Amplification (TLA) technology from Cergentis can be used to selectively amplify, sequence and phase the entire CFTR gene. With PacBio long-read SMRT Sequencing, TLA amplicons are sequenced intact and long-range phasing information of all fragments in entire amplicons is retrieved. Experimental Design and Methods: The TLA process produces amplicons consisting of 5-10 proximity ligated DNA fragments. TLA was performed on cell line and genomic DNA from Coriell GM12878, which has few heterozygous SNVs in CFTR, and the IB3 cell line, with known haplotypes but heterozygous for the delta508 mutation. All sample types were prepared with high and low density TLA primer sets, targeting coverage of >100 kb of the CFTR gene. Conclusion: We have demonstrated the power and utility of TLA with long-read SMRT Sequencing as a valuable research tool in sequencing and phasing across very long regions of the human genome. This process can be done in an efficient manner, multiplexing multiple genes and samples per SMRT Cell in a process amenable to high-throughput sequencing.

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