Informatics on High-Throughput Sequencing Data

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CBW HT-seq Integrative Assignment

Written originally by Mathieu Bourgey, edited by Florence Cavalli

Task

We will perform the same analysis as in Module 3 but using the mother and father samples i.e sample NA12891 and NA12891.

The fastq files are in the following directory of the cloud instance: ~/CourseData/HT_data/Module3/

 * raw_reads/NA12891/NA12891_CBW_chr1_R1.fastq.gz
 * raw_reads/NA12891/NA12891_CBW_chr1_R2.fastq.gz
 * raw_reads/NA12892/NA12892_CBW_chr1_R1.fastq.gz
 * raw_reads/NA12892/NA12892_CBW_chr1_R2.fastq.gz

Environment setup

#set up
export SOFT_DIR=/usr/local/
export WORK_DIR=~/workspace/HTseq/Integrative_Assignment/
export TRIMMOMATIC_JAR=$SOFT_DIR/Trimmomatic-0.36/trimmomatic-0.36.jar
export PICARD_JAR=$SOFT_DIR/picard/picard.jar
export GATK_JAR=$SOFT_DIR/gatk-4.0.1.2/gatk-package-4.0.1.2-local.jar
export GATK_OLD_JAR=~/CourseData/HT_data/software/GenomeAnalysisTK-3.8/GenomeAnalysisTK.jar
export BVATOOLS_JAR=~/CourseData/HT_data/software/bvatools-1.6/bvatools-1.6-full.jar
export REF=$WORK_DIR/reference/


rm -rf $WORK_DIR
mkdir -p $WORK_DIR
cd $WORK_DIR
ln -s ~/CourseData/HT_data/Module3/* .

Task list:

  1. Check read QC

  2. Trim unreliable bases from the read ends

  3. Align the reads to the reference

  4. Sort the alignments by chromosome position

  5. Realign short indels

  6. Fixe mate issues (optional)

  7. Mark duplicates

  8. Recalibrate the Base Quality

  9. Generate alignment metrics

Discussion/Questions:

  1. Explain the purpose of each step

  2. Which software tool can be used for each step

The full commands can be downloaded here solution