These algorithms generally use bayesian rules that compute the probability of a variant's occurring at a specific position while taking into account the known polymorphism rate and sequencing errors. The next step is to identify sequence variations from reference with specialized algorithms. On completion of sequence alignment, SAM (sequence alignment maps) or BAM (binary format) files are generated and imported into genome browsers such as IGV (Integrative Genomics Viewer) 42 from which they can be visualized. Non-gapped aligners, such as MAP and Bowtie, are best suited for the detection of insertions-deletions (indels). 39-41 Detection of variants is optimally achieved with gapped aligners, such as BWA and Novoalign. Whereas each platform typically provides an overall quality assessment, additional tools such as FastQC may be complementarily implemented.Īccurate alignment of sequence reads to reference requires implementation of algorithms such as BWA (Burrows-Wheeler Alignment), MAQ (Mapping and Assembly with Quality), Bowtie, and Novoalign. 37,38 Because of a tendency for deterioration of base quality at the ends of reads, trimming protocols are implemented to improve the quality of the data. In general, the different sequencing platforms use a parameter such as a Phred-like score, which is related logarithmically to the probabilities of the base-calling errors. Base-calling algorithms are implemented to reduce systematic errors. In general, the primary data outputs from each platform typically consist of text files containing sequence reads and the quality scores for each base. Nevertheless, several programs now exist to simplify NGS data analysis by provision of easy-to-use graphical interfaces. Many of the analytical programs still require command line computer languages and can be difficult for bioinformatics non-experts to use. Jaffe MD, in Hematopathology, 2017 Bioinformatics and Computational Methods for Next-Generation Sequencingīioinformatic analysis remains a challenge and bottleneck in the interpretation of NGS data.