May 1, 2017  |  General

SMRT Sequencing Enables Accurate Calling of Pathogenic Variants in Medically Relevant Genes

Screening for pathogenic variants associated with polycystic kidney disease is now more accurate and affordable with SMRT Sequencing. A new paper in Human Mutation from scientists at Leiden University Medical Center and other institutes reports the evaluation of long-read PacBio sequencing as a potential replacement for costly, time-consuming Sanger pipelines.
Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing” comes from lead author Daniel Borràs, senior author Seyed Yahya Anvar, and collaborators. The team notes that previous efforts to get away from conventional tools by implementing short-read sequencing were never successful enough for clinical use. “A genetic diagnosis of autosomal dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC-content, and homology of the PKD1 gene with six pseudogenes,” the scientists explain. In earlier studies, ambiguities from short-read sequencing “produced low true positive variant detection rates of 28% to 50% for the duplicated region of PKD1, and many false positives (10%) due to misalignments, low quality alignments and contamination by residual amplification of pseudogenes.”
The team predicted that long-read sequencing could address these issues, and evaluated the technology on 19 previously analyzed samples. They designed long-range PCR products to cover the coding regions of PKD1 and PKD2, and used PacBio’s Long Amplicon Analysis tool to reconstruct alleles from reads 3 kb or longer. Results were compared to those obtained previously from Sanger sequencing (requiring laborious long-range PCR, followed by many nested PCR reactions) and multiplex ligation-dependent probe amplification (MLPA). An initial examination of coverage found that “all PKD1 and PKD2 exons … from 19 ADPKD patients could be completely covered using long-reads.”
Variants detected with SMRT Sequencing and other approaches were compared; scientists determined that 17 high-confidence variants were detected by PacBio but not by Sanger. PacBio sequencing missed one pathogenic insertion, resulting in accurate calls for 18 of the 19 samples tested. “This provided a diagnosis for 94.7% of the patients, resulting in the correct detection of all PKD1 substitutions, single-nucleotide deletions, large deletions, one deletion-insertion, and 3 out of 4 insertions or duplications,” the scientists report.
These results point to SMRT Sequencing as an excellent replacement for older technologies to scan PKD1 and other medically relevant genes for pathogenic variants. “On top of reducing the PCR amplification steps required and limiting the implicit PCR artifacts, single molecule sequencing improves sequence alignments and aids in discriminating between homologous or repeated sequences, such as PKD1 pseudogenes,” the scientists write. “This provides a cleaner dataset for variant calling.”
The scientists conclude, “This method is highly valuable for a diagnostic setting, as it increases the resolution power of clinically relevant but difficult to sequence or to resolve genomic regions.”
Senior author Anvar from Leiden University Medical Center will be presenting at the SMRT Leiden events taking place this week. Follow along at #SMRTLeiden!

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