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This section provides directions for generating SSCS (Single Strand Consensus Sequence) reads and trimming molecular indexes from raw fastq files.
Learning Objectives:
- Use python script to generate SSCS Reads.
- Use flexbar to trim molecular indexes from duplex seq libraries.
- Use python script to generate SSCS Reads.
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Tutorial: SSCS Reads
Since this will take longer we will first use First we want to generate SSCS reads where we take advantage of the molecular indexes added during library prep. For the purpose of this tutorial, the paired end sequencing of sample DED110 has been placed in the $BI/gva_course/mixed_population directory. To do so we will use a "majority rules" python script (named SSCS_DCS.py) which was heavily modified by DED from a script originally created by Mike Schmitt and Scott Kennedy for the original duplex seq paper. This script can be found in the $BI/scripts directory. Invoking the script is as simple as typing SSCS_DCS.py; adding -h will give a list of the available options. The goal of this command is to generate SSCS reads, for any molecular index where we have at least 2 reads present, and to generate a log file which will tell us some information about the data.
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The following arguments are the ones that are needed to generate just the SSCS reads
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This should take ~15 minutes to complete in an idev shell. While this is completing we will generate .fastq files where the molecular index has been trimmed from the read.
Tutorial (Trimmed Reads):
For the purpose of this tutorial, we will be working with flexbar which like breseq is something that we have installed in the BioITeam as it is not a tacc module. Some additional modules must be loaded in order for it to work correctly, and the LD_LIBRARY_PATH variable must be modified as listed below.
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The following arguments are the ones that are needed to successfully trim the first 16 bases of the sequence:
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the trimmed_1/2.fastq is the trimmed fastq files the trimmed_1/2.fastq.lengthdist is the length distribution file (which should have 2 lines: a header line and a line showing that all of the reads are 85 bp long now the trimmed_1_single.fastq is the error file which should be empty |
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step:
You should now have 3 new .fastq files which we will use to call variants in: DED110_SSCS.fastq, trimmed_1.fastq, and trimmed_2.fastq. We will now run breseq to compare the number and quality of variants called.