[rbagnall]

i keep getting this error after ~20mins. Any idea?

Thanks,
Jason


java NGSrich -r 32815T_GATKrealigned_duplicates_marked.sam -g hg19 -t 0247401_D_BED_20090724_hg19.bed -T ngsrich_tmp -o ngsrich_out


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

READS FILE:
/mnt/Storage/All_Users/lij/testNGSrich/32815T_GATKrealigned_duplicates_marked.sam was reduced to /mnt/Storage/All_Users/lij/testNGSrich/ngsrich_tmp/1306393150286/NGSrich_32815T_GATKrealigned_duplicates_marked_pmc-bioinf02.14020.txt
Reduced file /mnt/Storage/All_Users/lij/testNGSrich/ngsrich_tmp/1306393150286/NGSrich_32815T_GATKrealigned_duplicates_marked_pmc-bioinf02.14020.txt sorted


java.io.FileNotFoundException: /mnt/Storage/All_Users/lij/testNGSrich/ngsrich_tmp/1306393150286/refGene.genome (No such file or directory)
at java.io.FileInputStream.open(Native Method)
at java.io.FileInputStream.<init>(FileInputStream.java:106)
at java.util.Scanner.<init>(Scanner.java:636)
at adapter.GenomeAdapter.adapt(GenomeAdapter.java:22)
at Enrichment.reduceFiles(Enrichment.java:181)
at NGSrich.main(NGSrich.java:91)
Exception in thread "main" java.lang.NullPointerException
at adapter.GenomeAdapter.adapt(GenomeAdapter.java:33)
at Enrichment.reduceFiles(Enrichment.java:181)
at NGSrich.main(NGSrich.java:91)

I get the same error too.

Did you solve the problem Jason?

[PeteH]

I get the same error too.

Did you solve the problem Jason?

Check that the reference annotation was successfully downloaded by NGSrich. In my case it was unable to download the annotation (due to firewall?) but once I manually downloaded the file and used the option -g <local_path_to_manually_downloaded_annotation> instead the program worked.

[rbagnall]

Check that the reference annotation was successfully downloaded by NGSrich. In my case it was unable to download the annotation (due to firewall?) but once I manually downloaded the file and used the option -g <local_path_to_manually_downloaded_annotation> instead the program worked.

Awesome! Thanks PeteH, got it to work.

Nice tool but I would add to others comments that it would be good to enable .bam files too

[chariko]

Dear All,

I would like to announce the inception of NGSrich, a software for evaluation of target enrichment performance in Illumina next-generation sequencing. An early release of the code has been uploaded to SourceForge at

http://sourceforge.net/projects/ngsrich/files/

but we're still working on a Java version. Regards,

Peter Frommolt
University of Cologne

Dear Peter,

I have run 5 Illumina samples with your program in and when I obtained the summary statistics I always obtain 2 reads more than the ones I obtain while doing a custom command in Linux. As I have written in my sam file "ILLUMINA" for every read, I do not understand why does NGSrich give me not the same number of reads.

grep -c ILLUMINA file

Example:
NGSrich: # Reads: 10754356
grep -c ILLUMINA myfile: 10754358

Can you help me? Thanks in advance

[rbagnall]

Hey chariko,

Sometimes a sam header contains the PL:ILLUMINA (i.e. platform) in the read group.

If you show the first 10 lines of your grep'ed' output, is there an @RG.... line?

Dear Peter,

I have run 5 Illumina samples with your program in and when I obtained the summary statistics I always obtain 2 reads more than the ones I obtain while doing a custom command in Linux. As I have written in my sam file "ILLUMINA" for every read, I do not understand why does NGSrich give me not the same number of reads.

grep -c ILLUMINA file

Example:
NGSrich: # Reads: 10754356
grep -c ILLUMINA myfile: 10754358

Can you help me? Thanks in advance

[chariko]

Hey chariko,

Sometimes a sam header contains the PL:ILLUMINA (i.e. platform) in the read group.

If you show the first 10 lines of your grep'ed' output, is there an @RG.... line?

Hi rbagnall,

First of all thank you for your answer but unfortunately I checked that and all the lines before the reads did not have any "ILLUMINA" written. As an example the first 30 lines of my file were:

@HD VN:1.0 SO:coordinate
@SQ SN:chr1 LN:135534747
@SQ SN:chr11 LN:135006516
@SQ SN:chr12 LN:133851895
@SQ SN:chr13 LN:115169878
@SQ SN:chr14 LN:107349540
@SQ SN:chr15 LN:102531392
@SQ SN:chr16 LN:90354753
@SQ SN:chr17 LN:81195210
@SQ SN:chr18 LN:78077248
@SQ SN:chr19 LN:59128983
@SQ SN:chr1 LN:249250621
@SQ SN:chr20 LN:63025520
@SQ SN:chr21 LN:48129895
@SQ SN:chr22 LN:51304566
@SQ SN:chr2 LN:243199373
@SQ SN:chr3 LN:198022430
@SQ SN:chr4 LN:191154276
@SQ SN:chr5 LN:180915260
@SQ SN:chr6 LN:171115067
@SQ SN:chr7 LN:159138663
@SQ SN:chr8 LN:146364022
@SQ SN:chr9 LN:141213431
@SQ SN:chrM LN:16571
@SQ SN:chrX LN:155270560
@SQ SN:chrY LN:59373566
@PG ID:bwa PN:bwa VN:0.5.9-r16
ILLUMINA-GA_0032:6:94:16...

Thanks

[zee]

Nice tool, some suggestions

1) Support for BAM files
2) Packaged program as a jar file
3) Ability to cap mapping quality
4) Ability to ignore reads flagged as duplicates (see SAM spec)
5) Consider a padding option for flanking N bases around a target region

[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

[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...

[pfrommolt]

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

[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

[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!!!

[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

[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 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