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PL-Grid instance of Galaxy provides all the tools necessary for quality control of your raw reads, using FASTQC software, and  for for preparation to alignment to the reference throughout trimming and filtering, using Flexbar software. This tutorial will give you an introduction to how to use these tools and it will guide you through the process.

Input Dataset

In this tutorial we  we will use the file ‘test_dataset.fastq’ which is available for download from the Bismark homepage (it contains 10,000 reads in FastQ format, Phred33 qualities, 50 bp long reads, from a human directional BS-Seq library).

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We could observe that "Per base sequence quality" figure (to the left) is better in comparison to the figure before trimming. Some of the reads with quality lower than 20 were excluded from the analysis, especially that at the end of reads (46-50 base pair). Similar changes can also be seen at "Sequence length (bp)" figure before and after trimming (to the right). The main part of reads remained without trimming (50 bp) but from some of them adapters (probably those which lower than 45 bp) and reads which have poor quality (46-49 bp) were cut off. The middle figure, "Per base sequence content" shows the mean percentage of nucleotides (A, C, G, T). We could observe that after trimming, mainly of adapters which have repetitive sequences, there was equalization of the mean nucleotide percentage in comparison to reads before trimming.

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