The use of technical replication for detection of low-level somatic mutations in next-generation sequencing

Kim, Junho; Kim, Dachan; Lim, Jae Seok; Maeng, Ju Heon; Son, Hyeonju; Kang, Hoon Chul; Nam, Hojung; Lee, Jehee; Kim, Sang Woo

doi:10.1038/s41467-019-09026-y

Cited by 54 publications

(60 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We aligned raw sequences from Fastq files to the hg19/GRCh37 assembly of the human genome reference sequence using BWA-MEM ( http://bio-bwa.sourceforge.net ). After recommended preprocessing, indel realignment, and base recalibration, we utilized RePlow ( https://sourceforge.net/projects/replow/ ), a variant caller designed to detect low-level somatic variants from replicated sample data [ 13 ]. We then applied in-house filtering criteria: 1) excluding registered common variants in a public database (common dbSNP147; https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg38&g=snp147Common ); 2) excluding variants with a putative low snpEFF impact score ( http://snpeff.sourceforge.net/SnpEff.html#intro ); 3) excluding variants with a PolyPhen & SIFT ≠ Damaging, phastCons score < 0.9; and 4) excluding variants with an allele frequency > 0.1% in the ExAC and gnomAD databases (both from https://gnomad.broadinstitute.org ) for minor allele frequencies of general and East Asian populations [ 14 ].…”

Section: Methodsmentioning

confidence: 99%

Detailed analysis of phenotypes and genotypes in megalencephaly-capillary malformation-polymicrogyria syndrome caused by somatic mosaicism of PIK3CA mutations

Park

Shin

Yoo

et al. 2020

Orphanet J Rare Dis

Self Cite

View full text Add to dashboard Cite

Background: Megalencephaly-capillary malformation-polymicrogyria syndrome (MCAP) belongs to a group of conditions called the PIK3CA-related overgrowth spectrum (PROS). The varying phenotypes and low frequencies of each somatic mosaic variant make confirmative diagnosis difficult. We present 12 patients who were diagnosed clinically and genetically with MCAP. Genomic DNA was extracted mainly from the skin of affected lesions, also from peripheral blood leukocytes and buccal epithelial cells, and target panel sequencing using high-depth nextgeneration sequencing technology was performed. Results: Macrocephaly was present in 11/12 patients (92%). All patients had normal body asymmetry. Cutaneous vascular malformation was found in 10/12 patients (83%). Megalencephaly or hemimegalencephaly was noted in all 11 patients who underwent brain magnetic resonance imaging. Arnold-Chiari type I malformation was also seen in 10 patients. Every patient was identified as having pathogenic or likely pathogenic variants of the PIK3CA gene. The variant allele frequency (VAF) ranged from 6.3 to 35.3%, however, there was no direct correlation between VAF and the severity of associated anomalies. c.2740G > A (p.Gly914Arg) was most commonly found, in four patients (33%). No malignancies developed during follow-up periods. Conclusions: This is the first and largest cohort of molecularly diagnosed patients with MCAP in Korea. Targeted therapy with a PI3K-specific inhibitor, alpelisib, has shown successful outcomes in patients with PROS in a pilot clinical study, so early diagnosis for genetic counseling and timely introduction of emerging treatments might be achieved in the future through optimal genetic testing.

show abstract

Section: Methodsmentioning

confidence: 99%

Detailed analysis of phenotypes and genotypes in megalencephaly-capillary malformation-polymicrogyria syndrome caused by somatic mosaicism of PIK3CA mutations

Park

Shin

Yoo

et al. 2020

Orphanet J Rare Dis

Self Cite

View full text Add to dashboard Cite

show abstract

“…The gold standard for BRAF mutation analysis is molecular biology (DNA-based) techniques, with classical Sanger sequencing being the most commonly used [41,42]. However, in the case of CNS tumors, in particular ganglioglioma, the relevance of the latter method has recently been questioned [40,43], due to its high detection threshold of 20% allele frequency, which does not work well with a low number and/or scattered distribution of neoplastic cells [44]. Although other molecular biology techniques, such as pyrosequencing, next-generation sequencing, PNA-clamping PCR, ddPCR, and ASqPCR, have higher sensitivity (detection of 0.02-10% mutant in a background of wild type), all DNA-based methods are often expensive, labor-intensive, time-consuming, and not widely available in pathology laboratories, also because they require a highly skilled operator and complex infrastructure [45,46].…”

Section: Discussionmentioning

confidence: 99%

Detection of BRAF V600E Mutation in Ganglioglioma and Pilocytic Astrocytoma by Immunohistochemistry and Real-Time PCR-Based Idylla Test

Durślewicz

Klimaszewska‐Wiśniewska

Antosik

et al. 2020

Disease Markers

View full text Add to dashboard Cite

The BRAF V600E mutation is an important oncological target in certain central nervous system (CNS) tumors, for which a possible application of BRAF-targeted therapy grows continuously. In the present study, we aim to determine the prevalence of BRAF V600E mutations in a series of ganglioglioma (GG) and pilocytic astrocytoma (PA) cases. Simultaneously, we decided to verify whether the combination of fully automated tests—BRAF-VE1 immunohistochemistry (IHC) and Idylla BRAF mutation assay—may be useful to accurately predict it in the case of specified CNS tumors. The study included 49 formalin-fixed, paraffin-embedded tissues, of which 15 were GG and 34 PA. Immunohistochemistry with anti-BRAF V600E (VE1) antibody was performed on tissue sections using the VentanaBenchMark ULTRA platform. All positive or equivocal cases on IHC and selected negative ones were further assessed using the Idylla BRAF mutation assay coupled with the Idylla platform. The BRAF-VE1 IHC was positive in 6 (6/49; 12.3%) and negative in 39 samples (39/49; 79.6%). The interpretation of immunostaining results was complicated in 4 cases, of which 1 tested positive for the Idylla BRAF mutation assay. Therefore, the overall positivity rate was 14.3%. This included 2 cases of GG and 5 cases of PA. Our study found that BRAF V600E mutations are moderately frequent in PA and GG and that for these tumor entities, IHC VE1 is suitable for screening purposes, but all negative, equivocal, and weak positive cases should be further tested with molecular biology techniques, of which the Idylla system seems to be a promising tool.

show abstract

“…The output is typically in the Variant Call Format (VCF) so as to be represented in structured text-based formats. 17 The RePlow method can detect somatic variants by estimations of the background and variant models for a given observation. Furthermore, to determine high-confidence variants, we performed some filtering steps based on depths (total depth ≥15, alternative allele depth ≥5) and functional impact (e.g., synonymous or nonsynonymous).…”

Section: Discussionmentioning

confidence: 99%

“…16 To detect somatic mutations among all high-confidence variants, we used the RePlow method that jointly analyzes library-level replicates for accurate detection of low-VAF somatic mutations. 17 The RePlow method can detect somatic variants by estimations of the background and variant models for a given observation. Copy number alterations (CNAs) were called by the CNV detection tool of the GATK4 beta version.…”

Section: Discussionmentioning

confidence: 99%

Genomic profiling of the residual disease of advanced high‐grade serous ovarian cancer after neoadjuvant chemotherapy

Kim

Shim

Bae

et al. 2019

Intl Journal of Cancer

Self Cite

View full text Add to dashboard Cite

The goal of our study was to demonstrate the spectrum of genomic alterations present in the residual disease of patients with advanced high-grade serous ovarian cancer (HGSOC) after neoadjuvant chemotherapy (NAC), including matched pretreatment biopsies. During the study period between 2006 and 2017, we collected pre-NAC and post-NAC tumor tissue samples from patients with advanced HGSOC. We performed combined next-generation sequencing and immunohistochemistry to identify actionable targets and pathway activation in post-NAC residual tumors. We also examined whether post-NAC profiling of residual HGSOC identified targetable molecular lesions in the chemotherapy-resistant component of tumors. Among 102 post-NAC samples, 41 (40%) of patients had mutations in homologous recombination repair (HRR) genes (HRR deficiency). Patients with HRR mutations had higher tumor mutation burdens (p < 0.001) and higher alterations in the PI3K-AKT-mTOR pathway (p = 0.004) than patients without these HRR mutations. Nevertheless, we found no significant differences in progression-free survival (p = 0.662) and overall survival (OS; p = 0.828) between the two groups. Most patients (91%) had alterations in at least one of the targetable pathways, and those patients with cell cycle (p = 0.004) and PI3K-AKT-mTOR signaling (p = 0.005) pathway alterations had poorer OS (Bonferroni-corrected threshold = 0.0083, 0.05/6). We showed the genomic landscape of tumor cells remaining in advanced HGSOC after NAC. Once validated, these data can help inform biomarker-driven adjuvant studies in targeting residual tumors to improve the outcomes of patients with advanced HGSOC after NAC.

show abstract

The use of technical replication for detection of low-level somatic mutations in next-generation sequencing

Cited by 54 publications

References 37 publications

Detailed analysis of phenotypes and genotypes in megalencephaly-capillary malformation-polymicrogyria syndrome caused by somatic mosaicism of PIK3CA mutations

Detailed analysis of phenotypes and genotypes in megalencephaly-capillary malformation-polymicrogyria syndrome caused by somatic mosaicism of PIK3CA mutations

Detection of BRAF V600E Mutation in Ganglioglioma and Pilocytic Astrocytoma by Immunohistochemistry and Real-Time PCR-Based Idylla Test

Genomic profiling of the residual disease of advanced high‐grade serous ovarian cancer after neoadjuvant chemotherapy

Contact Info

Product

Resources

About