FAQ

Questions:
1. Do you have sample data and its results obtained from the Genomon-exome pipeline?
2. Can't find expected mutations.
3. How to update Copy Number analysis tools?

Q1. Do you have sample data and its results obtained from the Genomon-exome pipeline?

Yes, we provide the sample FASTQ data and the result files. To find them follow the instructions below:

# change directory to the input dir. You need to mkdir if not.

		# change directory to the input dir
		cd exome/data/input
		# Get the sample
		wget http://genomon.hgc.jp/data/exome/Simulation.tar.gz
		# Unpack the input FASTQ file.
		tar xzvf Simulation.tar.gz
		
		# change directory to the db dir
		cd exome/db 
		# Get the interval list for Simulation
		wget http://genomon.hgc.jp/data/exome/interval_list_hg19_simulation.tar.gz
		# Unpck the interval list.
		tar xzvf interval_list_hg19_simulation.tar.gz
		
		# change directory to the script dir
		cd exome/script
		# do the alignment - Command 1, Alignment & Generate Summary Table 
		qsub map_bwa.sh Simulation 120917 SureSelect50M  # arg3 should be the .bed file describes capture regions
		# do the realignment - Command 3, Realignment 
		qsub realign_gatk.sh Simulation 120917 normal tumor -i interval_list_hg19_simulation
		# Command 4, Mutation Calling (Fisher's Exact Text) & Annotation - non realigned bams
		qsub fisher_test.sh Simulation 120917_aligned normal tumor -t 120917 -g map_bwa -f ga.bam -i interval_list_hg19_simulation
		# Command 4, Mutation Calling (Fisher's Exact Text) & Annotation - realigned bams
		qsub fisher_test.sh Simulation 120917_realigned normal tumor -t 120917 -i interval_list_hg19_simulation
		# Note that the sample data should use only for Commands 1, 3, and 4

Results can be obtained through the links below. It is recommended to open them using Excell on your PC.

Results with Not realigned bam files.
sum_Simulation_120917_aligned_tumor.exome.result.txt.gz
sum_Simulation_120917_aligned_tumor.genome.result.txt.gz
Results with realigned bam files.
sum_Simulation_120917_realigned_tumor.exome.result.txt.gz
sum_Simulation_120917_realigned_tumor.genome.result.txt.gz

The Summary
summary.tar.gz

Q2. Can't find expected mutations.

You might want to check the bam file with the Integrative Genomics Viewer, IGV viewer. It is suggested to launch IGV directly on one of the Supercomputing nodes.

Step 1. Get IGV. You need to register at the Homepage before you download. After registration, you'll be guided to the download page. Copy the link address to the binary, IGV_(version).zip and download the binary from the link.

		# GET IGV 
		wget "link to IGV_(version).zip"
		# UNPACK IGV
		unzip IGV_(version).zip

Step 2. Configure FreeNX, please see: How to use FreeNX .

Step 3. Launch IGV. You logon to the Supercomputer via FreeNX. After you logon, fire up a Konsole terminal. Then type:

		# launch IGV
		bash ${dir IGV unpacked}/IGV_(version)/igv.sh

Step 4. Load the bam file. Check the mutation of your interest exists in the bam file.

Case A: Mutation can't be seen in the bam file
Mapping was failed. Check the sample set again to make sure it was the sample you needed to look at. Copy number data analysis might help you with this. Remember, if no alignment there should not be a mutation call.

Case B: Mutation is seen in the bam file.
Check the mapping and base qualities of the mutation candidates. If qualiy values are low, it is possible that the mutation were filtered out in the steps Mutation Calling (Fisher's Exact Text) & Annotation.
You can chage the both quality values in Mutation Calling (Fisher's Exact Text) & Annotation, please vary the values and see if you can find it.

		# If you want to do set; mapping quality = 20 and base quality = 10
		qsub fisher_test.sh -m 20 -b 10 sample01 120430 normal tumor
		# Defaults are; mapping quality = 30 and base quality = 15
		# Lower the filter, you get more false positives
		# optimized values vary sample to sample. 

Q3. How to update Copy Number analysis tools?

Get the Genomon-exome pakcage on to your PC via https://github.com/Genomon/exome_for_HGC/downloads . You can obtain either the .zip or .tar.gz archive. The file name looks something like this:

Genomon-exome_for_HGC-1.0-1-gc831ea8.zip

Find the copy_number directory under the directory you just unpacked the archive and upload it to the Supercomputer. You can place it under the Genomon-exome package directory.
If you aren't sure where to upload, consult Directory Structure and upload the copy_number dir in to the equivalent directory hierarchy.

If you have questions not answered on this FAQ page, please contact us.