[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

[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

[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,

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

[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?

[@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

[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

[ulz_peter]

I actually never solved the problem, but I will look into that next week. Anyone else had that problem solved already?

[Lien]

Oh, I just thought that because you sent your BED-file that you knew afterwards what was wrong.
I'll keep on searching then, but if anyone else knows how to fix this, please let us know.

Thanks!

[ulz_peter]

just tried it once more with the new version but still no data for some chromosomes...

[@abdallah]

Hi ulz_peter and Lien,

could u please check, if the reduced target and read files (names of the files starts with "NGSrich") in the temporary folder are both sorted by the reference name (e.g. chromosome) in the lexicographical order? (something like: chr1, chr10, ..., chr2, chr21, chr22, chr3, chr4, ..., chr9, chrX, chrY, ..).

I would also like to see, how u start the program.

Regards
Ali

[brentp]

First, thanks for making this tool available, it looks like it will be very useful.

I am attempting to use this on very large BAM files, it is creating a lot of large temporary files.

Is the .sam file actually needed, or is it just used to create the .txt file? If it's not needed, you could add the following to the bam2sam.sh

samtools view $BAM | awk 'BEGIN {OFS="\t"}{ print $1, $3, $4, $4 + length($10) - 1 }'

to get your .txt file.

Also, if your .BAM file is known to be sorted, then that will already be sorted, so the sort step is unnecessary, it would be nice if it were an option like --assume-sorted to further reduce the extra file processing.

Generally, I see the sort invocation as -k3,3n but you have -k3,n3. Out of curiosity, is there a difference.

[ulz_peter]

Hi Ali,

Yes the files are lexicographically ordered.
I invoked it as

java NGSrich evaluate -r GH1.bam.finished.bam -u hg19 -t ~/RefSeq/refGene_hg19_280510/RefGene_hg19.bed -s GH1

[Lien]

My reference genome and the BAM-file are lexicographically ordered. (I even tried a new reference genome with novel alignment using BWA).

My command: java NGSrich evaluate -r sample1.bam -u hg19 -t SureSelect.bed -o /NGSrich/sample1

Still don't know where it is going wrong.

Lien

[Lien]

I do get a warning during step 3 as follows:

Warning message:
In sortchr(levels(as.factor(bedfile$V1))) : NAs introduced by coercion
Created plots and HTML summary report

STEP 3 successfully completed