1. Dowell Short Read Class Phillip Richmond
ReSequencing
Dowell Short Read Class
Phillip Richmond

2. Outline The Plan Organize and copy data to your own working directory
Map reads back to a reference genome
Convert sam to bam
Remove duplicates
Run a variant caller
Visualize variants

3. Plan
The first round of variant calling we’re going to do will involve cutting the yeast genome Sigma1278b into reads, mapping them back to the S288c reference genome, and then finding all SNP differences between the two genomes
This data will be synthetic
The reads will already be produced for you in fastq format, 1x50 bp reads

4. Getting started Organization is KEY!!
For the resequencing tutorial this is the organization that will be necessary:
Make a new directory in your home directory called: ReSequencing
Inside of ReSequencing make subdirectories:
GENOME
FASTQ
SAM
VCF
PBS

5. Copying the data Now we want to copy the data from:
/projects/sreadgrp/homeworkfiles/ReSequencing/
Copy the Fastq file from the FASTQ directory (Sigmav7_50mers.fastq) to your own FASTQ directory
Copy SGDv4.fasta from GENOME/ to your own directory GENOME/
Copy the PBS files to your own PBS directory:
IndexGenome.pbs
MapReads.pbs
Sam2Bam.pbs
IndelRealign.pbs
CallSNPs.pbs

6. Index the genome (IndexGenome.pbs)
Command:
/opt/bowtie/bowtie /bowtie2-build <in.fasta> <out_index>
My Command:
/opt/bowtie/bowtie /bowtie2-build /Users/richmonp/ReSequencing/GENOME/SGDv4.fasta /Users/richmonp/ReSequencing/GENOME/SGDv4_bowtie2_Index

7. Map the reads back to the genome (MapReads.pbs)
These reads need to have “readgroups” in order to work.
It’s best to add these when we map using the bowtie2 options --rg and --rg-id:
Example:
--rg-id Sigmav7vsS288c_bowtie2 –rg SM:Sigmav7vsS288c_bowtie2
Full Command:
/opt/bowtie/bowtie /bowtie2 --rg-id Sigmav7vsS288c_bowtie2 --rg SM:Sigmav7vsS288c_bowtie2 /Users/richmonp/ReSequencing/GENOME/SGDv4_bowtie2_Index /Users/richmonp/ReSequencing/FASTQ/Sigmav7_50mers.fastq –S /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.sam 2> /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.stderr

8. Convert your file format using Samtools (Sam2Bam.pbs)
samtools view –bS <in.sam> -o <out.bam>
samtools sort <in.bam> <out.sorted>
samtools index <in.sorted.bam>
/opt/samtools/0.1.18/samtools view –bS /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.sam –o /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.bam
/opt/samtools/0.1.18/samtools sort /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.bam /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.sorted
/opt/samtools/0.1.18/samtools index /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.sorted.bam

9. Samtools remove duplicates (Sam2Bam.pbs)
Removes duplicate reads from PCR errors in reads.
samtools rmdup <in.sorted.bam> <out.rmdup.sorted.bam>
/opt/samtools/0.1.18/samtools rmdup /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2.sorted.bam /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup.sorted.bam

10. Realign around indels (IndelRealign.pbs)
GATK has a two-step process for realigning reads around indels
Step 1: Find candidate locations that may be best represented by an insertion or deletion
GATK’s RealignerTargetCreator
Step 2: Apply local realignment around the candidate locations to produce a new bam file
GATK’s IndelRealigner

11. Realign around Indels: RealignerTargetCreator
java –jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar –R <reference genome> -T RealignerTargetCreator (options) –I <in.sorted.rmdup.bam> -o <out.intervals>
java -jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar -R /Users/richmonp/ReSequencing/GENOME/SGDv4.fasta \
-T RealignerTargetCreator -minReads 5 \
-I /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup.sorted.bam
-o /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup.intervals

12. Realign around indels: IndelRealigner
java –jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar –T IndelRealigner –model USE_READS –targetIntervals <in.intervals> -R <reference.fasta> -I <in.rmdup.sorted.bam> -o <out.rmdup.realigned.sorted.bam>
java -jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar -T IndelRealigner -model USE_READS \
-targetIntervals /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup.intervals \
-R /Users/richmonp/ReSequencing/GENOME/SGDv4.fasta \
-I /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup.sorted.bam
-o /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup_realigned.sorted.bam

13. Call variants using GATK UnifiedGenotyper (CallSNPs.pbs)
The GATK package is a java executable, or a .jar file.
To run the package you type:
java –jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar
Then you must select a –T, or a program within the package to run, which in our case is UnifiedGenotyper
java –jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar –T UnifiedGenotyper

14. Call variants using GATK UnifiedGenotyper
java –jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar –T UnifiedGenotyper -glm BOTH -I <in.sorted.bam> -R <in.fasta> -o <out.vcf>
java -jar /opt/gatk/2.4-9/GenomeAnalysisTK.jar -T UnifiedGenotyper -glm BOTH -R /Users/richmonp/ReSequencing/GENOME/SGDv4.fasta -I /Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup_realigned.sorted.bam -o /Users/richmonp/ReSequencing/VCF/Sigmav7_vs_S288c_bowtie2_gatk.vcf

15. View your VCF in IGV
GATK automatically indexes your VCF files, so now we can visualize both the reads and SNPs in IGV
Transfer both the final bam file (/Users/richmonp/ReSequencing/SAM/Sigmav7_vs_S288c_bowtie2_rmdup_realigned.sorted.bam) and the vcf file (/Users/richmonp/ReSequencing/VCF/Sigmav7_vs_S288c_bowtie2_gatk.vcf) to your student directory on /projects/sreadgrp/student/<username>/
Open up the visualization VNC window
Open IGV
Load the files

16. Organize into groups of 5
Coffee Break
Then…
Organize into groups of 5

17. Paired-end data
The main difference between paired-end and single-end data will occur when you are mapping
Each read in the pair is denoted by either “R1” or “R2”
1028_S1_L001_R1_001.fastq
1028_S1_L001_R2_001.fastq

18. How it changes your bowtie2 command:
Open up MapPairedReads.pbs in an editor
Notice:
-1 /data/Avery/FASTQ/1056_S1_L001_R1_001.fastq \
-2 /data/Avery/FASTQ/1056_S1_L001_R2_001.fastq \
The -1 is for read 1, and the -2 is for read 2

19. Now…
Copy the MapPairedReads.pbs to your own PBS directory (from /projects/sreadgrp/homeworkfiles/ReSequencing/PBS/)
Copy a pair of fastq files to your FASTQ directory (only copy the ones based on your group problem sheet)

20. First group to map, call variants, and visualize variants, wins
First group to map, call variants, and visualize variants, wins! (prizes are not amazing)