Analytical and Potential Clinical Performance of Oncomine Myeloid Research Assay for Myeloid Neoplasms.

INTRODUCTION: Next-generation sequencing (NGS) panels have recently been introduced to efficiently detect genetic variations in hematologic malignancies. OBJECTIVES: Our aim was to evaluate the performance of the commercialized Oncomine™ myeloid research assay (OMA) for myeloid neoplasms. METHODS: Certified reference materials and clinical research samples were used, including 60 genomic DNA and 56 RNA samples. NGS was performed using OMA, which enables the interrogation of 40 target genes, 29 gene fusions, and five expression target genes with five expression control genes by the Ion S5 XL Sequencer. The analyzed data were compared with clinical data using karyotyping, reverse transcription polymerase chain reaction (PCR), fluorescence in situ hybridization, Sanger sequencing, customized NGS panel, and fragment analysis. RESULTS: All targets of reference materials were detected except three (two ASXL1 and one CEBPA) mutations, which we had not expected OMA to detect. In clinical search samples, OMA satisfactorily identified DNA variants, including 90 single nucleotide variants (SNVs), 48 small insertions and deletions (indels), and eight FLT3 internal tandem duplications (ITDs) (Kappa agreement 0.938). The variant allele frequencies of SNVs and indels measured by OMA correlated well with clinical data, whereas those of FLT3-ITDs were significantly lower than with fragment analysis (P = 0.008). Together, OMA showed strong ability to identify RNA gene fusions (Kappa agreement 0.961), except one RUNX1-MECOM. The MECOM gene was highly expressed in all five samples with MECOM-associated rearrangements, including inv(3), t(3;3), and t(3;21). CONCLUSION: OMA revealed excellent analytical and potential clinical performance and could be a good replacement for conventional molecular tests.

KRGDB: the large-scale variant database of 1722 Koreans based on whole genome sequencing.

Since 2012, the Center for Genome Science of the Korea National Institute of Health (KNIH) has been sequencing complete genomes of 1722 Korean individuals. As a result, more than 32 million variant sites have been identified, and a large proportion of the variant sites have been detected for the first time. In this article, we describe the Korean Reference Genome Database (KRGDB) and its genome browser. The current version of our database contains both single nucleotide and short insertion/deletion variants. The DNA samples were obtained from four different origins and sequenced in different sequencing depths (10× coverage of 63 individuals, 20× coverage of 194 individuals, combined 10× and 20× coverage of 135 individuals, 30× coverage of 230 individuals and 30× coverage of 1100 individuals). The major features of the KRGDB are that it contains information on the Korean genomic variant frequency, frequency difference between the Korean and other populations and the variant functional annotation (such as regulatory elements in ENCODE regions and coding variant functions) of the variant sites. Additionally, we performed the genome-wide association study (GWAS) between Korean genome variant sites for the 30×230 individuals and three major common diseases (diabetes, hypertension and metabolic syndrome). The association results are displayed on our browser. The KRGDB uses the MySQL database and Apache-Tomcat web server adopted with Java Server Page (JSP) and is freely available at http://coda.nih.go.kr/coda/KRGDB/index.jsp. Availability: http://coda.nih.go.kr/coda/KRGDB/index.jsp.

BRCA1/2 somatic mutation detection in formalin-fixed paraffin embedded tissue by next-generation sequencing in Korean ovarian cancer patients.

INTRODUCTION: The detection of BRCA1/2 mutations is important because PARP1 inhibitors are approved for germline and/or somatic BRCA-mutated advanced ovarian cancer. Next-generation sequencing (NGS) is increasingly used in clinical practice for BRCA1/2 mutations. The purpose of this study was to consider several conditions of NGS BRCA1/2 assay applicable to clinical laboratory tests, in particular for using formalin fixed paraffin embedded (FFPE) ovarian tissues. MATERIALS AND METHODS: We selected 64 ovarian cancer patients and performed Oncomine™ BRCA assay using FFPE tissue. Effect of FFPE sample quality was analyzed by NGS quality parameters including deamination metric. Somatic variants were selected by removing germline variants of peripheral blood and interpreted as pathogenic, variants of unknown significance, and false positive. RESULTS: We found a positive relationship between the number of variants over the deamination metric and FFPE age (P < 0.001) with a cutoff values of approximately 0.7 and 60 months, respectively. When comparing NGS results with Sanger sequencing, NGS misreported 3 of 15 variants using default parameters which were corrected after changing parameters. We detected somatic variants in eight patients and classified them into pathogenic (n = 3), VUS (n = 3) and false positive (n = 2). CONCLUSIONS: This study is important for improving BRCA1/2 mutation detection capabilities of NGS analytical pipelines and strategy to overcome their limitations using FFPE tissue in ovarian cancer patients.

HIA: a genome mapper using hybrid index-based sequence alignment.

BACKGROUND: A number of alignment tools have been developed to align sequencing reads to the human reference genome. The scale of information from next-generation sequencing (NGS) experiments, however, is increasing rapidly. Recent studies based on NGS technology have routinely produced exome or whole-genome sequences from several hundreds or thousands of samples. To accommodate the increasing need of analyzing very large NGS data sets, it is necessary to develop faster, more sensitive and accurate mapping tools. RESULTS: HIA uses two indices, a hash table index and a suffix array index. The hash table performs direct lookup of a q-gram, and the suffix array performs very fast lookup of variable-length strings by exploiting binary search. We observed that combining hash table and suffix array (hybrid index) is much faster than the suffix array method for finding a substring in the reference sequence. Here, we defined the matching region (MR) is a longest common substring between a reference and a read. And, we also defined the candidate alignment regions (CARs) as a list of MRs that is close to each other. The hybrid index is used to find candidate alignment regions (CARs) between a reference and a read. We found that aligning only the unmatched regions in the CAR is much faster than aligning the whole CAR. In benchmark analysis, HIA outperformed in mapping speed compared with the other aligners, without significant loss of mapping accuracy. CONCLUSIONS: Our experiments show that the hybrid of hash table and suffix array is useful in terms of speed for mapping NGS sequencing reads to the human reference genome sequence. In conclusion, our tool is appropriate for aligning massive data sets generated by NGS sequencing.