Dataset Information

A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for EGFR Mutations in Plasma from Non-Small Cell Lung Cancer Patients.

ABSTRACT: Sel-Cap^TM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R, and T790M, and compared results to those of PNAclamp^TM tumor biopsy (reference method, a peptide nucleic acid-mediated polymerase chain reaction clamping) and two other NGS liquid biopsies. Over two-thirds of activating mutations (Ex19del and L858R), previously confirmed by PNAclamp, were detected by Sel-Cap, which is 4-5 times more sensitive than NGS liquid biopsy. Sel-Cap showed particularly high sensitivity for T790M (88%) and for early-stage plasma samples. The relationship between initial T790M detection in plasma and progression-free survival (PFS) following first-line EGFR-TKIs was evaluated in 34 patients. Patients with T790M detected at treatment initiation (±3 months) had significantly shorter PFS than patients where T790M was first detected >3 months post treatment initiation (median PFS: 5.9 vs. 26.5 months; p < 0.0001). However, time from T790M detection to disease progression was not significantly different between the two groups (median around 5 months). In conclusion, Sel-Cap is a highly sensitive platform for EGFR mutations in plasma, and the timing of the first appearance of T790M in plasma, determined via highly sensitive liquid biopsies, may be useful for prediction of disease progression of NSCLC, around 5 months in advance.

SUBMITTER: Shin JY

PROVIDER: S-EPMC7760633 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for EGFR Mutations in Plasma from Non-Small Cell Lung Cancer Patients.

Shin Jung-Young JY Kim Jeong-Oh JO Lee Mi-Ran MR Kim Seo Ree SR Beck Kyongmin Sarah KS Kang Jin Hyoung JH

Cancers 20201130 12

Sel-CapTM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R ...[more]

PMID: 33266057

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Project description:Assessment of genomic DNA sequence variation and genotype calling in autotetraploids implies the ability to distinguish among five possible alternative allele copy number states. This study demonstrates the accuracy of genotyping-by-sequencing (GBS) of a large collection of autotetraploid potato cultivars using next-generation sequencing. It is still costly to reach sufficient read depths on a genome wide scale, across the cultivated gene pool. Therefore, we enriched cultivar-specific DNA sequencing libraries using an in-solution hybridisation method (SureSelect). This complexity reduction allowed to confine our study to 807 target genes distributed across the genomes of 83 tetraploid cultivars and one reference (DM 1-3 511). Indexed sequencing libraries were paired-end sequenced in 7 pools of 12 samples using Illumina HiSeq2000. After filtering and processing the raw sequence data, 12.4 Gigabases of high-quality sequence data was obtained, which mapped to 2.1 Mb of the potato reference genome, with a median average read depth of 63× per cultivar. We detected 129,156 sequence variants and genotyped the allele copy number of each variant for every cultivar. In this cultivar panel a variant density of 1 SNP/24 bp in exons and 1 SNP/15 bp in introns was obtained. The average minor allele frequency (MAF) of a variant was 0.14. Potato germplasm displayed a large number of relatively rare variants and/or haplotypes, with 61% of the variants having a MAF below 0.05. A very high average nucleotide diversity (??=?0.0107) was observed. Nucleotide diversity varied among potato chromosomes. Several genes under selection were identified. Genotyping-by-sequencing results, with allele copy number estimates, were validated with a KASP genotyping assay. This validation showed that read depths of ?60-80× can be used as a lower boundary for reliable assessment of allele copy number of sequence variants in autotetraploids. Genotypic data were associated with traits, and alleles strongly influencing maturity and flesh colour were identified.

Project description:Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

Dataset Information

A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for EGFR Mutations in Plasma from Non-Small Cell Lung Cancer Patients.

Publications

A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for <i>EGFR</i> Mutations in Plasma from Non-Small Cell Lung Cancer Patients.

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