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Hybrid capture-based targeted sequencing is increasingly used for genomic variants profiling in tumor patients. Unique molecular index (UMI) technology has recently been developed and helps to increase the accuracy of variant calling by minimizing PCR biases and sequencing errors. However, UMI-adopted targeted sequencing data analysis is slightly different from the methods for other types of omics data, and its pipeline for variant calling is still being optimized in various studying groups for their own purpose. Due to this provincial usage range of tools, our group built an analysis pipeline for global application to many studies of targeted sequencing generated with different methods. First, we generated hybrid capture-based data using genomic DNA extracted from tumor tissues of colorectal cancer patients. Sequencing libraries were prepared, pooled together and an 8-plexed capture library was processed to the enrichment step before 150bp paired-end (PE) sequencing with Illumina Hiseq series. For the analysis, we evaluated several different tools published. We mainly focused on the compatibility of input and output of each tool. Finally, our laboratory built an analysis pipeline specialized for UMI-adopted data. Through this pipeline, we were able to estimate the even on-target rates and filtered consensus reads for more accurate variant calling. These results suggest the potential of our analysis pipeline in the precise examination of quality and efficiency of conducted experiments.
This article was published in the following journal.
Name: Genomics & informatics
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Screening techniques first developed in yeast to identify genes encoding interacting proteins. Variations are used to evaluate interplay between proteins and other molecules. Two-hybrid techniques refer to analysis for protein-protein interactions, one-hybrid for DNA-protein interactions, three-hybrid interactions for RNA-protein interactions or ligand-based interactions. Reverse n-hybrid techniques refer to analysis for mutations or other small molecules that dissociate known interactions.
Techniques of nucleotide sequence analysis that increase the range, complexity, sensitivity, and accuracy of results by greatly increasing the scale of operations and thus the number of nucleotides, and the number of copies of each nucleotide sequenced. The sequencing may be done by analysis of the synthesis or ligation products, hybridization to preexisting sequences, etc.
A multistage process that includes RNA cloning, physical mapping, subcloning, sequencing, and information analysis.
A multistage process that includes DNA cloning, physical mapping, subcloning, sequencing, and information analysis.
A method of chemical analysis based on the detection of characteristic radionuclides following a nuclear bombardment. It is also known as radioactivity analysis. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. During DNA sequencing, the bases of a small fragment of DNA are sequentially identified from signals emitted as each fragment is re-synthesized from a ...
Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...