![]() ![]() While two of the reads are a perfect match to the reference, the three other reads show a mismatch each, highlighted in red ("A" in the read, instead of "T" in the reference). You can see the reference sequence on the top row, and five short reads stacked below this is called a pileup. GCTGATGTGCCGCCTCACTTCGGTGGTGAGGTG Reference sequence Reads aligned (mapped) to a reference sequence will look like this: A mapping algorithm will try to locate a (hopefully unique) location in the reference sequence that matches the read, while tolerating a certain amount of mismatch to allow subsequence variation detection. This is achieved by comparing the sequence of the read to that of the reference sequence. ![]() The line should have the same length as line 2, as there is one quality score per base.įor each of the short reads in the FASTQ file, a corresponding location in the reference sequence (or that no such region exists) needs to be determined. The scores are generated by the sequencing machine, and encoded as ASCII (33+score) characters. The quality scores of the bases from line 2.Today, this line is present for historical reasons backwards compatibility only. In very old FASTQ files, this is followed by the read name from the first line. The name/ID of the read, preceded by a For read pairs, there will be two entries with that name, either in the same or a second FASTQ file.Next-generation sequencing generally produces short reads or short read pairs, meaning short sequences of four lines are: There are certain instances (such as new genes in the sequenced sample that are not found in the existing reference sequence) that can not be detected by alignment alone however, while other approaches, such as de novo assembly, are potentially more powerful, they are also much harder or, for some organisms, impossible to achieve with current sequencing methods. Alignments of data from these re-sequenced organisms is a relatively simple method of detecting variation in samples. Having sequenced an organism of a species before, and having constructed a reference sequence, re-sequencing more organisms of the same species allows us to see the genetic differences to the reference sequence, and, by extension, to each other. Alignment, also called mapping, of reads is an essential step in re-sequencing. ![]()
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