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QC III: Library composition

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Bioinformatics Core Facility Gothenburg edited this page Jan 22, 2019 · 5 revisions

Course: HT17 Analysis-of-next-generation-sequencing-data (SC00024)

With FastQ Screen you can check that your libraries contain the genomes that are supposed to have, along with PhiX, Vectors or other contaminants commonly seen in sequencing experiments.

To run fastq_screen we need to determine which aligner will be used as well as which databases (or organisms) we want to use. For this exercise we will be using bowtie2 as aligner and Human, Mouse, PhiX and Leishmania as genomes to scan our samples. The Human and Mouse genomes are already in the server together with their corresponding bowtie2 indexes, ready to use! However, we will practice on how to set the PhiX and the Leishmania databases with 2 different approaches.

Create a directory called db, follow the following steps within that directory:

  • PhiX:

    1. iGenomes are a collection of reference sequences and annotation files for commonly analyzed organism.

    2. Go to the webpage and download the PhiX - Illumina - RTA build with wget webaddress

    3. Uncompress it with tar -xvzf file

    4. Check that the bowtie2 index is at PhiX/Illumina/RTA/Sequence/Bowtie2Index/genome

  • Leishmania

    1. Ensembl is a huge source for genomic data (among others).

    2. Find your way to the Leishmania infantum JPCM5 webpage in Ensembl

    3. Download Leishmania_infantum_jpcm5.ASM287v2.dna.toplevel.fa.gz which contains all chromosomes for this protist with wget webaddress

    4. Build the bowtie2 index using bowtie2-build -h

    5. Check that *bt2 files are created

Now that we have all the data needed, we need to tell fastq_screen where it can find the indexes. Copy the fastq_screen.conf.example from /home/teacher3/doctos. Have a look and modify the following:

  • Set the BOWTIE2 line to /usr/local/bin/bowtie2, uncomment the line

  • Set the THREADS 8 line to 1, though this make things quicker, we may overload the server!

  • Set the DATABASE Human to: see links to db document in GU, uncomment the line

  • Set the DATABASE Mouse to: see links to db document in GUL, uncomment the line

  • Set the DATABASE PhiX to /your_home_directory/db/PhiX/Illumina/RTA/Sequence/Bowtie2Index/genome, uncomment the line

  • Add a new line for Leishmania so it looks like:

    DATABASE<tab>Leishmania<tab>/your_home_directory/db/Leishmania_infantum_jpcm5.ASM287v2.dna.toplevel.fa

Create a directory called FastQScreen and save the following results of your samples there.

fastq_screen has not been installed globally, so type $fastq_screen, this variable contains the path where the tool was installed

Once you are done, you can run multiqc on the FastQScreen to gather your output files, or look at the original files.

Q. Do you have any contamination in your samples? Does the composition of the samples make sense?

Next: QC IV: Quality filtering

Previous: QC II: Quality check

Created by Marcela Dávila

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