Browsing by Subject "eksomisekvensointi"

The next-generation sequencing (NGS) platforms create a large amount of sequence in short amount of time, when compared to first generation sequencers. An overview of the NGS platforms is provided with more in-depth look into Illumina Genome Analyzer II as that is used to create the data for the thesis. There were two main aims in this thesis. First, to create a pipeline which can be used to analyse genomic sequencing. Second, to use the pipeline to compare whole human exome capture methods from two manufacturers, Roche Nimblegen and Agilent. The pipeline is describe in detail in material and methods. All the inputs for the pipeline are described and examples shown. In the pipeline the given sequences are first aligned against the reference genome. Then various separate analysis is performed to retrieve variants and coverage of the sequencing. Supplementary results include paired-end anomalies, larger insertion and deletion polymorphisms and assembly of non-aligned sequences. The two capture methods are also described and changes to the manufacturers' recommended protocols are listed. Finally, the section has the options and various inputs used in the pipeline runs of the exome data. The results of the pipeline is a basic level of analysis of the sequencing as well as various graphs showing the quality of the run. All the output files intended for user are described. By using the results of the pipeline, the user can do more in-depth analysis as required by the project. When comparing the two exome capture methods, the Nimblegen capture was shown to be more efficient in capturing the CCDS exome. While the Agilent capture kit provided better one fold coverage over the exome, higher fold coverage (over 10 fold), which is required for reliable variant calling in nextgeneration sequencing, was better reached using the Nimblegen capture kit. Also, significantly fewer false positive paired-end anomalies were observed in the library created by using the Nimblegen capture.