Large empty data using Depthofcoverage

Question

[mauro.castagnetta]

Dear all,

I'm trying to use DepthofCoverage tool on a bam file deriving from (IonTorrent obtained) fastq aligned using bwa.

I'm using my .refseq file which is converted from .bed file and it contains my genes of interest, they are 12 genes with several exons.

I'm obtaining after about 72h of analysis (on a core i7 and 16 gb of ram) only a huge sequence of zeros, zero intervals, zero coverage etc. etc.

Can anyone give me some advice?

Thanks in advance,

M

[Irsan]

Do you mean the DepthOfCoverage-walker of the Genome analysis tool kit (GATK)? If so, it helps when you give the exact command you used and it helps even more when you have any log-messages.

[mauro.castagnetta]

Yes, I was referring to DepthOfCoverage-walker from gatk2. I'm using it under Galaxy..

This is what I obtain from logs:

	INFO 14:18:37,907 HelpFormatter - --------------------------------------------------------------------------------
	INFO 14:18:37,908 HelpFormatter - The Genome Analysis Toolkit (GATK) v2.8-1-g932cd3a, Compiled 2013/12/06 16:47:15
	INFO 14:18:37,908 HelpFormatter - Copyright (c) 2010 The Broad Institute
	INFO 14:18:37,908 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk
	INFO 14:18:37,910 HelpFormatter - Program Args: -T DepthOfCoverage --num_threads 1 --calculateCoverageOverGenes /home/mauro/galaxy/galaxy-dist/database/files/000/dataset_443.dat --partitionType sample --partitionType readgroup --partitionType library --out /home/mauro/galaxy/galaxy-dist/database/files/000/dataset_449.dat --outputFormat csv --maxBaseQuality 127 --maxMappingQuality 2147483647 --minBaseQuality -1 --minMappingQuality 127 --nBins 499 --start 1 --stop 500 -I /home/mauro/galaxy/galaxy-dist/database/tmp/tmp-gatk-UVueP7/gatk_input_0.bam -R /home/mauro/galaxy/galaxy-dist/database/tmp/tmp-gatk-UVueP7/gatk_input.fasta
	INFO 14:18:37,910 HelpFormatter - Date/Time: 2015/04/15 14:18:37
	INFO 14:18:37,910 HelpFormatter - --------------------------------------------------------------------------------
	INFO 14:18:37,910 HelpFormatter - --------------------------------------------------------------------------------
	INFO 14:18:38,306 GenomeAnalysisEngine - Strictness is SILENT
	INFO 14:18:38,374 GenomeAnalysisEngine - Downsampling Settings: No downsampling
	INFO 14:18:38,379 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
	INFO 14:18:38,404 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.01
	INFO 14:18:38,847 GenomeAnalysisEngine - Preparing for traversal over 1 BAM files
	INFO 14:18:38,910 GenomeAnalysisEngine - Done preparing for traversal
	INFO 14:18:38,910 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]
	INFO 14:18:38,910 ProgressMeter - Location processed.sites runtime per.1M.sites completed total.runtime remaining
	INFO 14:19:08,912 ProgressMeter - chr1:422625 1.97e+05 30.0 s 2.5 m 0.0% 59.5 h 59.5 h
	INFO 14:20:08,913 ProgressMeter - chr1:1161081 1.16e+06 90.0 s 77.0 s 0.0% 66.6 h 66.6 h
	INFO 14:21:08,914 ProgressMeter - chr1:1849573 1.79e+06 2.5 m 83.0 s 0.1% 70.0 h 70.0 h
	INFO 14:22:08,915 ProgressMeter - chr1:2515013 1.95e+06 3.5 m 107.0 s 0.1% 72.3 h 72.2 h
	INFO 14:23:18,916 ProgressMeter - chr1:3376061 3.13e+06 4.7 m 89.0 s 0.1% 71.9 h 71.8 h
	INFO 14:24:18,916 ProgressMeter - chr1:4213589 4.23e+06 5.7 m 80.0 s 0.1% 70.0 h 69.9 h
	INFO 14:25:18,917 ProgressMeter - chr1:4892641 4.69e+06 6.7 m 85.0 s 0.2% 71.0 h 70.9 h
	INFO 14:26:18,918 ProgressMeter - chr1:5580617 5.32e+06 7.7 m 86.0 s 0.2% 71.6 h 71.5 h
	INFO 14:27:28,919 ProgressMeter - chr1:6395153 6.21e+06 8.8 m 85.0 s 0.2% 72.0 h 71.9 h
	INFO 14:28:28,919 ProgressMeter - chr1:7096477 6.80e+06 9.8 m 86.0 s 0.2% 72.3 h 72.1 h
	INFO 14:29:28,920 ProgressMeter - chr1:7789301 7.80e+06 10.8 m 83.0 s 0.2% 72.6 h 72.4 h
	INFO 14:30:28,921 ProgressMeter - chr1:8480301 7.80e+06 11.8 m 91.0 s 0.3% 72.8 h 72.6 h
	INFO 14:31:38,921 ProgressMeter - chr1:9288941 9.19e+06 13.0 m 84.0 s 0.3% 73.0 h 72.8 h
	INFO 14:32:38,922 ProgressMeter - chr1:9977373 9.57e+06 14.0 m 87.0 s 0.3% 73.2 h 73.0 h
	INFO 14:33:38,923 ProgressMeter - chr1:10676113 1.01e+07 15.0 m 88.0 s 0.3% 73.3 h 73.1 h
	INFO 14:34:38,924 ProgressMeter - chr1:11378237 1.11e+07 16.0 m 86.0 s 0.4% 73.4 h 73.2 h
	INFO 14:35:48,924 ProgressMeter - chr1:12197225 1.21e+07 17.2 m 85.0 s 0.4% 73.5 h 73.2 h
	INFO 14:36:48,925 ProgressMeter - chr1:12897809 1.29e+07 18.2 m 84.0 s 0.4% 73.6 h 73.2 h
	INFO 14:37:48,926 ProgressMeter - chr1:13799273 1.37e+07 19.2 m 84.0 s 0.4% 72.5 h 72.2 h
	INFO 14:38:58,927 ProgressMeter - chr1:14608637 1.40e+07 20.3 m 87.0 s 0.5% 72.7 h 72.4 h
	INFO 14:40:08,927 ProgressMeter - chr1:15387057 1.53e+07 21.5 m 84.0 s 0.5% 73.0 h 72.6 h
	INFO 14:41:08,928 ProgressMeter - chr1:16079369 1.56e+07 22.5 m 86.0 s 0.5% 73.1 h 72.7 h
	INFO 14:42:08,929 ProgressMeter - chr1:16781565 1.67e+07 23.5 m 84.0 s 0.5% 73.1 h 72.8 h
	INFO 14:43:18,930 ProgressMeter - chr1:17651313 1.75e+07 24.7 m 84.0 s 0.6% 73.0 h 72.6 h
	INFO 14:44:28,930 ProgressMeter - chr1:18464493 1.83e+07 25.8 m 84.0 s 0.6% 73.1 h 72.7 h
	INFO 14:45:28,931 ProgressMeter - chr1:19149205 1.85e+07 26.8 m 86.0 s 0.6% 73.2 h 72.8 h
	INFO 14:46:28,932 ProgressMeter - chr1:19830605 1.85e+07 27.8 m 90.0 s 0.6% 73.3 h 72.9 h
	INFO 14:47:38,933 ProgressMeter - chr1:20637805 1.85e+07 29.0 m 93.0 s 0.7% 73.4 h 72.9 h
	INFO 14:48:48,933 ProgressMeter - chr1:21418733 2.13e+07 30.2 m 85.0 s 0.7% 73.6 h 73.1 h
	INFO 14:49:48,934 ProgressMeter - chr1:22101017 2.17e+07 31.2 m 86.0 s 0.7% 73.7 h 73.2 h
	INFO 14:50:48,935 ProgressMeter - chr1:22794621 2.26e+07 32.2 m 85.0 s 0.7% 73.7 h 73.2 h
	INFO 14:51:48,936 ProgressMeter - chr1:23487921 2.26e+07 33.2 m 87.0 s 0.7% 73.8 h 73.2 h
	INFO 14:52:58,936 ProgressMeter - chr1:24295045 2.40e+07 34.3 m 85.0 s 0.8% 73.8 h 73.3 h
	INFO 14:53:58,937 ProgressMeter - chr1:24988701 2.46e+07 35.3 m 86.0 s 0.8% 73.9 h 73.3 h
	INFO 14:54:58,938 ProgressMeter - chr1:25681437 2.55e+07 36.3 m 85.0 s 0.8% 73.9 h 73.3 h
	INFO 14:56:08,939 ProgressMeter - chr1:26491357 2.64e+07 37.5 m 85.0 s 0.8% 74.0 h 73.3 h
	INFO 14:57:18,939 ProgressMeter - chr1:27304869 2.67e+07 38.7 m 86.0 s 0.9% 74.0 h 73.4 h
	INFO 14:58:18,940 ProgressMeter - chr1:28010969 2.67e+07 39.7 m 89.0 s 0.9% 74.0 h 73.3 h
	INFO 14:59:18,941 ProgressMeter - chr1:28716377 2.85e+07 40.7 m 85.0 s 0.9% 74.0 h 73.3 h
	INFO 15:00:28,942 ProgressMeter - chr1:29542377 2.85e+07 41.8 m 88.0 s 0.9% 74.0 h 73.3 h
	INFO 15:01:28,942 ProgressMeter - chr1:30400349 2.99e+07 42.8 m 86.0 s 1.0% 73.6 h 72.9 h
	INFO 15:02:28,943 ProgressMeter - chr1:31103873 3.09e+07 43.8 m 85.0 s 1.0% 73.6 h 72.9 h
	INFO 15:03:28,944 ProgressMeter - chr1:31806741 3.13e+07 44.8 m 85.0 s 1.0% 73.7 h 72.9 h
	INFO 15:04:38,944 ProgressMeter - chr1:32620741 3.13e+07 46.0 m 88.0 s 1.0% 73.7 h 72.9 h
	INFO 15:05:38,945 ProgressMeter - chr1:33323201 3.32e+07 47.0 m 84.0 s 1.1% 73.7 h 72.9 h
	INFO 15:06:38,946 ProgressMeter - chr1:33996225 3.38e+07 48.0 m 85.0 s 1.1% 73.8 h 73.0 h
	INFO 15:07:48,947 ProgressMeter - chr1:34781825 3.38e+07 49.2 m 87.0 s 1.1% 73.9 h 73.1 h
	INFO 15:08:48,947 ProgressMeter - chr1:35455325 3.38e+07 50.2 m 89.0 s 1.1% 73.9 h 73.1 h
	INFO 15:09:48,948 ProgressMeter - chr1:36131425 3.38e+07 51.2 m 90.0 s 1.2% 74.0 h 73.2 h
	INFO 15:10:48,949 ProgressMeter - chr1:36804129 3.67e+07 52.2 m 85.0 s 1.2% 74.1 h 73.2 h
	INFO 15:11:58,949 ProgressMeter - chr1:37590245 3.76e+07 53.3 m 85.0 s 1.2% 74.2 h 73.3 h
	INFO 15:12:58,950 ProgressMeter - chr1:38282185 3.81e+07 54.3 m 85.0 s 1.2% 74.2 h 73.3 h
	INFO 15:13:58,951 ProgressMeter - chr1:38978637 3.90e+07 55.3 m 85.0 s 1.2% 74.2 h 73.3 h
	INFO 15:15:08,952 ProgressMeter - chr1:39793077 3.96e+07 56.5 m 85.0 s 1.3% 74.2 h 73.3 h
	INFO 15:16:08,953 ProgressMeter - chr1:40489749 4.01e+07 57.5 m 86.0 s 1.3% 74.2 h 73.3 h
	INFO 15:17:08,954 ProgressMeter - chr1:41184349 4.01e+07 58.5 m 87.0 s 1.3% 74.2 h 73.3 h
	INFO 15:18:08,955 ProgressMeter - chr1:41884749 4.01e+07 59.5 m 89.0 s 1.3% 74.2 h 73.3 h
	INFO 15:19:18,955 ProgressMeter - chr1:42698701 4.22e+07 60.7 m 86.0 s 1.4% 74.3 h 73.2 h
	INFO 15:20:18,956 ProgressMeter - chr1:43401857 4.28e+07 61.7 m 86.0 s 1.4% 74.3 h 73.2 h
	INFO 15:21:18,957 ProgressMeter - chr1:44104657 4.28e+07 62.7 m 87.0 s 1.4% 74.3 h 73.2 h
	INFO 15:22:18,964 ProgressMeter - chr1:44795957 4.28e+07 63.7 m 89.0 s 1.4% 74.3 h 73.2 h
	INFO 15:23:28,965 ProgressMeter - chr1:45607705 4.52e+07 64.8 m 86.0 s 1.5% 74.3 h 73.2 h
	INFO 15:24:28,967 ProgressMeter - chr1:46298073 4.60e+07 65.8 m 85.0 s 1.5% 74.3 h 73.2 h
	INFO 15:25:28,967 ProgressMeter - chr1:46996509 4.65e+07 66.8 m 86.0 s 1.5% 74.3 h 73.2 h
	INFO 15:26:28,968 ProgressMeter - chr1:47658109 4.65e+07 67.8 m 87.0 s 1.5% 74.4 h 73.3 h
	INFO 15:27:28,969 ProgressMeter - chr1:48311917 4.79e+07 68.8 m 86.0 s 1.5% 74.5 h 73.3 h
	INFO 15:28:38,969 ProgressMeter - chr1:49094517 4.79e+07 70.0 m 87.0 s 1.6% 74.5 h 73.4 h
	INFO 15:29:38,970 ProgressMeter - chr1:49769721 4.93e+07 71.0 m 86.0 s 1.6% 74.6 h 73.4 h
	INFO 15:30:38,971 ProgressMeter - chr1:50450921 4.93e+07 72.0 m 87.0 s 1.6% 74.6 h 73.4 h
	INFO 15:31:48,972 ProgressMeter - chr1:51224421 4.93e+07 73.2 m 89.0 s 1.6% 74.7 h 73.4 h
	INFO 15:32:48,972 ProgressMeter - chr1:51886921 4.93e+07 74.2 m 90.0 s 1.7% 74.7 h 73.5 h
	INFO 15:33:48,973 ProgressMeter - chr1:52577121 4.93e+07 75.2 m 91.0 s 1.7% 74.7 h 73.5 h
	INFO 15:34:48,974 ProgressMeter - chr1:53262549 5.28e+07 76.2 m 86.0 s 1.7% 74.7 h 73.5 h

view raw biostars-138981.txt hosted with ❤ by GitHub

and to clarify, the data I've obtained are here:

http://pastebin.com/cypy96Fc

(only a small part...continue for several GBs..)

Thanks!

Mauro

Answer 1

[Irsan]

It appears as if you haven't told the DepthOfCoverage tool to restrict the analysis to you genes of interest. So what happens is that you get depth of coverage for the complete genome which in your case is most likely 0 for most of the genome. In the log-file you provided you can see a list of paramaters that you have set for the analysis (after: Program Args:). There should be something like -L /path/to/yourfileofinterest or --intervals /path/to/yourfileofinterest. I think your first problem right now is how to a compatible (Picard-style) interval-list file. You can have a look in the GATK documentation for more information.