[pfrommolt]

If you have downloaded and unzipped the annotation from

http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz

(example for hg19), you can just specify the local path to this file instead of 'hg19'.

[husamia]

It would be nice to include example of report to see what types of metrics it can calculate. I am curious about this.

[NGSfan]

Hi! I got it to work, the output looks correct! It's strange though I can see Step 3 and Step 5 as unsuccessful, but the output looks finished (includes list of poor and high covered genes).

I really like the summary.



java NGSrich -r /net/ngs/NGS.analysis/data/091204_ILLUMINA-075005_0000_42GHRAAXX/s_1.42GHRAAXX_LIB001_MIAPACA2_24877_FID000.J00001.alnRecal.sam -a /net/ngs/NGS.analysis/bin/annovar/humandb/hg18_refGene.txt -t /net/ngs/NGS.analysis/reference/500_Target_Regions.bed -T /dev/shm/
test: /net/ngs/NGS.analysis/data/091204_ILLUMINA-075005_0000_42GHRAAXX/enrichment
=======================1=======================
>>> STEP 1: reducing files
READS FILE:
/net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast.sam was reduced to /dev/shm/1305908202090/NGSrich_s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_vieasncl6.20049.txt
Reduced file /dev/shm/1305908202090/NGSrich_s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_vieasncl6.20049.txt sorted

GENOME ANNOTATION FILE:
/dev/shm/1305908202090/hg18_refGene.genome reduced to /dev/shm/1305908202090/NGSrich_genome_vieasncl6.20049.txt

TARGET REGIONS FILE:
/dev/shm/1305908202090/1000plus_Target_Regions.target reduced to /dev/shm/1305908202090/NGSrich_target_vieasncl6.20049.txt
Reduced file /dev/shm/1305908202090/NGSrich_target_vieasncl6.20049.txt sorted

STEP 1 successfully completed

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

Starting computing target coverage files.
Mean target coverage data computed (/net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/enrichment/s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_enrichment.bed).
Detailed base coverage computed (/dev/shm/1305908202090/coverage_vieasncl6.20049.txt).
Coverage summary computed (/net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/enrichment/s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_enrichment.xml).

STEP 2 successfully completed

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

Detecting evaluation program
"data" and "plots" subdirectories created and xml and bed file moved to the data subdirectory!
Run evaluation program on the following arguments Writing HTML output ... ready.Finished.
HTML FILE /net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/enrichment/s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_enrichment.html not found378_0070_B81MCAABXX/enrichment/110415_enrichment.html
Creating coverage barplot ... ready.Searching for poorly (<2x) and highly (>200x) covered genes ...AABXX_LIB160_HT_26828_FID047_J00229.bfast_enrichment.bed
STEP 3 unsuccessfulbarplots ... ready.Creating coverage barplot ...CAABXX/enrichment
Creating coverage pieplot ... ready.Preparing coverage barplots ...efGene.txt
=====================4=========================eplot ...
>>> STEP 4: computing targets wiggle datale ...
R Ausgabe: Reading XML file ...
Start computing target-based wiggle data
Details File Name: /dev/shm/1305908202090/coverage_vieasncl6.20049.txt
Output Dir: /net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/enrichment/data
End of computing target-based wiggle data

STEP 4 successfully completed.

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

Start computing overall wiggle data
Align File Name: /dev/shm/1305908202090/NGSrich_s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_vieasncl6.20049.txt
Output Dir: /net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/enrichment/data
/net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/enrichment/s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast_enrichment.wignot found

STEP 5 unsuccessful

===============================================

[1]+ Done java NGSrich -r /net/ngs/NGS.analysis/data/110415_SN378_0070_B81MCAABXX/s_1.B81MCAABXX_LIB160_HT_26828_FID047_J00229.bfast.sam -a /net/ngs/NGS.analysis/bin/annovar/humandb/hg18_refGene.txt -t /net/ngs/NGS.analysis/reference/1000plus_Target_Regions.bed -T /dev/shm/

[dnusol]

Hi,

I am having a similar problem. It seems that the sam file and the database are fine but then I get this error

java NGSrich -r /Data/Exp1/s_3_QC_remdup.sam -a hg19 -T /Data/Exp1/ -t /Data/Exp1/exomeplusv2.bed
test: /Data/Exp1/enrichment
=======================1=======================
>>> STEP 1: reducing files

READS FILE:
/Data/Exp1/s_3_QC_remdup.sam was reduced to /Data/Exp1/1306423511007/NGSrich_s_3_QC_remdup_KDavid.26819.txt
Reduced file /Data/Exp1/1306423511007/NGSrich_s_3_QC_remdup_KDavid.26819.txt sorted

GENOME ANNOTATION FILE:
/Data/Exp1/1306423511007/refGene.genome reduced to /Data/Exp1/1306423511007/NGSrich_genome_KDavid.26819.txt
Exception in thread "main" java.lang.ArrayIndexOutOfBoundsException: Array index out of range: 1
at java.util.Vector.get(Vector.java:721)
at adapter.Adapter.field(Adapter.java:90)
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)

This is a head result of my .bed file

head /Data/Exp1/exomeplusv2.bed
track:exome
chr1 30275 30431
chr1 69069 70029
chr1 228233 228354
chr1 228471 228711
chr1 367647 368608
chr1 470971 471330
chr1 621084 622045
chr1 741165 741285
chr1 745438 745558

Any ideas?

Thanks,

Dave

[jtjli]

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)

[PeteH]

Hi Peter,

This looks like a very useful tool! Do you have plans to allow the input file to be in BAM rather than SAM format? Also, is it possible to include the median coverage along with the mean coverage?

Thanks,
Pete

[@abdallah]

Hi,

Hi,

I am having a similar problem. It seems that the sam file and the database are fine but then I get this error

java NGSrich -r /Data/Exp1/s_3_QC_remdup.sam -a hg19 -T /Data/Exp1/ -t /Data/Exp1/exomeplusv2.bed
test: /Data/Exp1/enrichment
=======================1=======================
>>> STEP 1: reducing files

READS FILE:
/Data/Exp1/s_3_QC_remdup.sam was reduced to /Data/Exp1/1306423511007/NGSrich_s_3_QC_remdup_KDavid.26819.txt
Reduced file /Data/Exp1/1306423511007/NGSrich_s_3_QC_remdup_KDavid.26819.txt sorted

GENOME ANNOTATION FILE:
/Data/Exp1/1306423511007/refGene.genome reduced to /Data/Exp1/1306423511007/NGSrich_genome_KDavid.26819.txt
Exception in thread "main" java.lang.ArrayIndexOutOfBoundsException: Array index out of range: 1
at java.util.Vector.get(Vector.java:721)
at adapter.Adapter.field(Adapter.java:90)
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)

This is a head result of my .bed file

head /Data/Exp1/exomeplusv2.bed
track:exome
chr1 30275 30431
chr1 69069 70029
chr1 228233 228354
chr1 228471 228711
chr1 367647 368608
chr1 470971 471330
chr1 621084 622045
chr1 741165 741285
chr1 745438 745558

Any ideas?

Thanks,

Dave

You should just remove the first line of the target file because it doesn't have the expected form (ie "chrome start end"). In the next version we will probably allow comments within the files.

Regards,
@abdallah

[@abdallah]

Hi,

@dnusol
You should remove the header from your .bed-file because it causes the parse error, since the program expects only lines with the following format (chrom start end). We will maybe allow comments or headers in the next versions.

Regards,
@abdallah

[Vanisha]

Hey this looks like a great tool - can it only be used for samtools/bwa alignments? what about novoalign?

[ulz_peter]

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!

[PeteH]

Hi
I tried to run NGSrich_0.4.3 on a SAM file generated using Novoalign using the command
java NGSrich -r ../../B6113_NMT5_Novoalign/novoalign_simple/sam/B6113_novoalign.sam -g mm9 -t ../../B6113_NMT5_Novoalign/capture_target.alpha.bed

I received the following error:

test: /usr/local/work/hickey/NGSrich_0.4.3/bin/../../B6113_NMT5_Novoalign/novoalign_simple/sam/enrichment
File /scratch/ccg-ngs/tmp/1308184671325/ not found
Program stopped.

Can you please help?

[ulz_peter]

I got the same error. It worked when I specified another tmp folder using the -T command (in my case -T /tmp)

[PeteH]

I got the same error. It worked when I specified another tmp folder using the -T command (in my case -T /tmp)

Thanks, got it running!

[pfrommolt]

Does this error show up in the output BED file already? This is located in the 'data' subdirectory and probably has only '0.0' coverage values for the chromosomes you mention. Could you pls email this BED file to me?

Regards,
Peter

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!

[ulz_peter]

Yes, the error shows up in the bed file. All the chromosomes which do not show any bars in the histogram have 0.0 values. Could you send a PM with your E-mail adress?