[Lien]

Hi,

NGSrich looks very useful, I've been searching for a tool like that for a long time now...

However, I've got some problems ( I use version 0.4.3). I did alignemnt using bwa and a reference sequence which has chromosomes ordered 1-22XYMT with no unplaced unocalized or alternative contigs. The graphs for the first chromosomes (chr1, chr10, chr11, chr12, chr13, chr14, chr16 and chr 17) but it seems to discard the rest of the data. I just get empty plots for the other chromosomes. I was thinking about using a reference file with the chromosomes in the order as stated in the Alignment.html file, but that doesn't work as we analyze our data using GATK (and GATK wants to have the reference sequence ordered...)

I started your program as: java NGSrich -r Alignment.sam -a hg19 -t refGene.bed -T /tmp

Is that a problem of the reference sequence or am I making something wrong.

Thank you for your help and the great tool.
P.S.: BAM Support would indeed facilitate use of your tool!

Hi all,

I just have the exact same problem that the NGSrich seems to discard some of the data (chr 2-9, X and Y).
As with you, the BED-file is empty for these chromosomes.
Could you tell me how you solved this problem? The tool seems very very useful, but I don't find any solution for this on the forum.

Thanks a lot!
Lien

[@abdallah]

Dear All,

We just uploaded the new version of NGSrich (v0.5.5). Here are the changes in the last three versions:

Version 0.5.5 (uploaded 2011-19-09)
-> Option "--no-details" added to repress the computation
of the details reports

Version 0.5.4 (uploaded 2011-09-09)
-> Fixed bug in the pre-sorting step
-> Reduced number of plots in the details reports

Version 0.5.3 (uploaded 2011-08-11)
-> Enrichment details for each target region
-> Several bugs fixed
-> More exceptions on erroneous handling by user

The software was tested on several test cases. We welcome new bug reports and suggestions for new or enhanced features.

Regards
Ali

[Vanisha]

I keep getting this error:


>>> Found BAM file: converting to SAM

=======================1=======================
>>> STEP 1: reducing files

Exception in thread "main" java.util.NoSuchElementException: No line found
at java.util.Scanner.nextLine(Unknown Source)
at adapter.readAdapter.SamAdapter.adapt(SamAdapter.java:127)
at Enrichment.reduceFiles(Enrichment.java:139)
at NGSrichEvaluate.main(NGSrichEvaluate.java:110)
at NGSrich.main(NGSrich.java:26)

my command is

java NGSrich evaluate -r /data_n2/vplagnol/Projects/exome_reseq/aligned_celiac_hg19/CAP152646_NgenInSol/CAP152646_NgenInSol_sorted_unique.bam -g h19 -t /data_n2/hmw208/seq_cap_nimblegenEXOME/references/080904_ccds_exome_rebalfocus_HX1.bed

Any idea?

[tonio100680]

Dear Peter,

For the first question : R is installed in another folder (/opt /R/) and NGSrich is installed in folder : /stockage/tools/NGSrich_0.4.5
For the second question : my webbroswer (mozilla firefox) does not display the file :
<SampleSet><NumberSamples>1</NumberSamples><Sample><SampleName>align_LBC03</SampleName><NumberReads>207164</NumberReads><TargetSize>39173</TargetSize><AvTargetCoverage>0.0</AvTargetCoverage><SDTargetCoverage>0.0</SDTargetCoverage><ReadsOnTarget><OnTarget><NumberReads>0</NumberReads><PercReads>0.0</PercReads></OnTarget><Plus100>
.....
<NumberBases>0</NumberBases><PercBases>0.0</PercBases></from30x></TargetCovered></Sample></SampleSet>

For the .wig files in UCSC browser :
Error File 'align_LBC03.wig' - track load error (track name='ct_align_4907'):
Couldn't find size of chromosome chr17.fa (note: chrom names are case sensitive)

Thanks

Antoine

[pfrommolt]

Dear Antoine,

first question: you need to have R installed and it has to be on your $PATH .
Second question: BED file can be opened using text editor, xml with webbroswer, wiggle files can be uploaded to a genome browser, e.g. at UCSC.

Best,
Peter

[tonio100680]

Dear Peter,
I am a beginner in the field of bioinformatics! How can I view my results in the "Data" (resulting files: align_LBC03_enrichment.bed align_LBC03_enrichment.xml align_LBC03_onTarget.wig align_LBC03.wig
).
I have to use R (ps: I do not have any files in the "plot")?
Thanks

[tonio100680]

Dear Peter,

Thanks for help !

I downloaded the new version. I use this command:

java NGSrich summarize -r /stockage/ngs-client-01/tools/samtools/test_target/sam/align_LBC03.sam -o /stockage/ngs-client-01/tools/samtools/test_target/sam/
Error: Argument -i is mandatory

Apparently missing the-i option

For this command :
java NGSrich evaluate -r /stockage/ngs-client-01/tools/samtools/test_target/sam/align_LBC03.sam -a /stockage/ngs-client-01/tools/samtools/test_target/ref/hg19_refGene.txt -t /stockage/ngs-client-01/tools/samtools/test_target/bed/baits_chr17_brca1.bed

I get this response:
>>> STEP 1: reducing files

READS FILE:
/stockage/ngs-client-01/tools/samtools/test_target/sam/align_LBC03.sam reduced to /tmp/1311070026218/NGSrich_align_LBC03_s-ngs-client-linux.10819.txt
Reduced file /tmp/1311070026218/NGSrich_align_LBC03_s-ngs-client-linux.10819.txt sorted

GENOME ANNOTATION FILE:
/tmp/1311070026218/hg19_refGene.genome reduced to /tmp/1311070026218/NGSrich_genome_s-ngs-client-linux.10819.txt

TARGET REGIONS FILE:
/tmp/1311070026218/baits_chr17_brca1.target reduced to /tmp/1311070026218/NGSrich_target_s-ngs-client-linux.10819.txt
Reduced file /tmp/1311070026218/NGSrich_target_s-ngs-client-linux.10819.txt sorted

STEP 1 successfully completed

=====================2=========================
>>> STEP 2: computing target coverage data

Target BED coverage file created (/stockage/ngs-client-01/tools/samtools/test_target/sam/enrichment/data/align_LBC03_enrichment.bed)
Per-base coverage file created (/tmp/1311070026218/coverage_s-ngs-client-linux.10819.txt)
Summary statistics file created (/stockage/ngs-client-01/tools/samtools/test_target/sam/enrichment/data/align_LBC03_enrichment.xml)

STEP 2 successfully completed

=====================3=========================
>>> STEP 3: evaluating enrichment files

java.io.IOException: Cannot run program "Rscript": java.io.IOException: error=2, No such file or directory
at java.lang.ProcessBuilder.start(ProcessBuilder.java:475)
at java.lang.Runtime.exec(Runtime.java:610)
at java.lang.Runtime.exec(Runtime.java:448)
at java.lang.Runtime.exec(Runtime.java:345)
at Enrichment.evaluate(Enrichment.java:179)
at NGSrichEvaluate.main(NGSrichEvaluate.java:122)
at NGSrich.main(NGSrich.java:26)
Caused by: java.io.IOException: java.io.IOException: error=2, No such file or directory
at java.lang.UNIXProcess.<init>(UNIXProcess.java:164)
at java.lang.ProcessImpl.start(ProcessImpl.java:81)
at java.lang.ProcessBuilder.start(ProcessBuilder.java:468)
... 6 more

=====================4=========================
>>> STEP 4: computing targets wiggle data

Computing wiggle file for on-target reads
Wiggle file for on-target reads created
Output written to /stockage/ngs-client-01/tools/samtools/test_target/sam/enrichment/data/align_LBC03_onTarget.wig

STEP 4 successfully completed.

=====================5=========================
>>> STEP 5: computing overall wiggle data

Computing wiggle file for all reads
Wiggle file for all reads created
Output written to /stockage/ngs-client-01/tools/samtools/test_target/sam/enrichment/data/align_LBC03.wig

STEP 5 successfully completed.

It's normal ?

I hold you informed of progress!

Regards
Antoine

[pfrommolt]

Dear tonio100680,

if you are using the most recent version (0.4.5), please note that the way the software is invoked has been changed (see README).
Also, please make use of the newly created mailing list for questions of any kind.

Best,
Peter

[tonio100680]

Hi,

My command :

[ngs@s-ngs-client-linux tools]$ java NGSrich -r /stockage/ngs-client-01/tools/samtools/test_target/sam/align_LBC03.sam -a /stockage/ngs-client-01/tools/samtools/test_target/ref/hg19_refGene.txt -t /stockage/ngs-client-01/tools/samtools/test_target/bed/baits_chr17_brca1.bed

Exception in thread "main" java.lang.NoClassDefFoundError: NGSrich
Caused by: java.lang.ClassNotFoundException: NGSrich
at java.net.URLClassLoader$1.run(URLClassLoader.java:217)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:205)
at java.lang.ClassLoader.loadClass(ClassLoader.java:319)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:294)
at java.lang.ClassLoader.loadClass(ClassLoader.java:264)
at java.lang.ClassLoader.loadClassInternal(ClassLoader.java:332)
Could not find the main class: NGSrich. Program will exit.

????

Regards

[pfrommolt]

Dear community,

I have just set up a mailing list for NGSrich users. Please subscribe at

https://lists.sourceforge.net/lists/listinfo/ngsrich-users

All best,
Peter

[scalabrin]

2 missing reads

>>> STEP 1: reducing files

READS FILE:
/path/tmp/1309943569477/sample10.sam was reduced to /path/tmp/1309943569477/NGSrich_sample10_pinot.5066.txt

in the second file there is one line less, the last one. Perhaps you forgot to parse the last line (after a while loop?). And a similar error might contribute to the two missing reads!!!

[scalabrin]

Bug and bam supported in new version

1) I got two reads less than expected (as chariko)
2) Mean coverage looks suspicious. At least in one sample where I expected about 50x coverage I got 501x coverage...
3) On target does not look correct for some samples (double than expected)

Wig files are not correctly computed in step 5 if underscores are present in the path (I checked with and without) but statistics do not change (conversely to what I supposed):

>>> STEP 5: computing overall wiggle data

Start computing overall wiggle data
Align File Name: /tmp_path/1309882900147/NGSrich_filename_server.20189.txt
Output Dir: /output_path/data
Overall conversion in Wig unsuccessful
java.io.FileNotFoundException: /output_path/data/sapiens/share/NGSrich/mydata/tmp_enrichment.wig (No such file or directory)
at java.io.FileOutputStream.open(Native Method)
at java.io.FileOutputStream.<init>(FileOutputStream.java:209)
at java.io.FileOutputStream.<init>(FileOutputStream.java:160)
at java.io.FileWriter.<init>(FileWriter.java:90)
at converters.Read2Wig.convert(Read2Wig.java:89)
at converters.Read2Wig.<init>(Read2Wig.java:65)
at Enrichment.computeOverallWiggleFile(Enrichment.java:368)
at NGSrich.main(NGSrich.java:127)

Indeed 'sapiens' directory is not present in 'data'. Is there any problem in parsing paths? In my path (output_path) there is a homo_sapiens directory, perhaps you do not take into account underscores?


I tried both latests versions 0.4.3 and 0.4.4.
Last version does work (indeed it is a simple but effective work-around calling samtools to produce a temporary sam file!)

ps I had to cut path names as they represent private data

[pfrommolt]

Dear community,
I just uploaded a new version (0.4.4) with BAM support. Please give it a try.
Best, Peter

[scalabrin]

Bugs

1) I got two reads less than expected (as chariko did)
2) Mean coverage looks suspicious. At least in one sample where I expected about 50x coverage I got 501x coverage...
3) On target does not look correct for some samples (about double than expected)

Point 1 happens at every experiment, points 2 and 3 just sometimes...

[m_elena_bioinfo]

Dear, I have a problem in step1 with NGSrich:
I run by this command:
> java NGSrich -r sample.sam -a hg19 -t BED_SureSelect_exon.bed -T /tmp/

and the program returns me:

READS FILE:
sample.sam was reduced to /tmp/1309850911320sample_genoma1.5988.txt
Reduced file /tmp/1309850911320
sample_genoma1.5988.txt sorted

GENOME ANNOTATION FILE:
/tmp/1309850911320/refGene.genome reduced to /tmp/1309850911320/NGSrich_genome_genoma1.5988.txt
Exception in thread "main" java.lang.NumberFormatException: For input string: "chr1"
at java.lang.NumberFormatException.forInputString(NumberFormatException.java:48)
at java.lang.Integer.parseInt(Integer.java:449)
at java.lang.Integer.parseInt(Integer.java:499)
at adapter.TargetAdapter.start(TargetAdapter.java:168)
at adapter.TargetAdapter.adapt(TargetAdapter.java:56)
at Enrichment.reduceFiles(Enrichment.java:185)
at NGSrich.main(NGSrich.java:91)

Could you help me?
Thanx a lot,
ME