[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

[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

[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]

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]

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

[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]

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?

[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?

[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!

[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!

[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]

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]

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!

[Vanisha]

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

[@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