[Jessica_L]

The vendor for my library kit is saying that they no longer use NGSrich (possibly because of the bugs that we're all reporting). They've switched to a combination solution of bedtools, samtools and Picard to calculate coverage uniformity and on-target bases.

[Jessica_L]

I'm trying to use NGSrich for the first time and I'm getting the same java errors that others are reporting.

Exception in thread "main" java.util.NoSuchElementException: No line found
at java.util.Scaner.nextLIne(Scanner.java:1540)
at _main.EnrichmentStatsComputer.ini(EnrichmentStatsComputer.java:287)
at _main.EnrichmentStatsComputer.<init>(EnrichmentStatsComputer.java:241)
at _main.Enrichment.computeTargetCoverageFiles(Enrichment.java:305)
at _main.NGSrichEvaluate.evaluate(NGSrichEvaluate.java:285)
at NGSrich.main(NGSrich.java:48)


Is there any kind of resolution for this error?

[doclevel]

Hi @abdallah ,
I have a problem when "computing target coverage data" (NGSrich v0.7.7)

java -Xmx40g -cp ../bin/NGSrich/ NGSrich evaluate -r analysis/G4_genome.sam -u hg19 -t ../Target/SeqCap_EZ_Exome_v3_capture.bed -a ../NGSrich_annotation/hg19_refGene.txt -s G4 -T ../tmp -o analysis/NGSrich
=======================1=======================
>>> STEP 1: reducing files
......
INFO 2012-09-03 16:10:18 MergeSamFiles 63000000 records read.
INFO 2012-09-03 16:10:35 MergeSamFiles 64000000 records read.
INFO 2012-09-03 16:10:52 MergeSamFiles 65000000 records read.
INFO 2012-09-03 16:10:56 MergeSamFiles Finished reading inputs.
GENOME ANNOTATION FILE:
../NGSrich_annotation/hg19_refGene.txt reduced to ../tmp/Sample_From_14_17_29_1346653049121/NGSrich_genome_HPC1.txt
TARGET REGIONS FILE:
/mafs1/data/project/exome/Project_12-048/../tmp/Sample_From_14_17_29_1346653049121/SeqCap_EZ_Exome_v3_capture.bed reduced to ../tmp/Sample_From_14_17_29_1346653049121/NGSrich_target_HPC1.bed

STEP 1 successfully completed

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

../tmp/Sample_From_14_17_29_1346653049121/G4_genome.sam
../tmp/Sample_From_14_17_29_1346653049121/NGSrich_target_HPC1.bed
../tmp/Sample_From_14_17_29_1346653049121/NGSrich_genome_HPC1.txt
Exception in thread "main" java.util.NoSuchElementException: No line found
at java.util.Scanner.nextLine(Scanner.java:1516)
at _main.EnrichmentStatsComputer.init(EnrichmentStatsComputer.java:287)
at _main.EnrichmentStatsComputer.<init>(EnrichmentStatsComputer.java:241)
at _main.Enrichment.computeTargetCoverageFiles(Enrichment.java:305)
at _main.NGSrichEvaluate.evaluate(NGSrichEvaluate.java:285)
at NGSrich.main(NGSrich.java:48)



And my target file is OK:
more ../Target/SeqCap_EZ_Exome_v3_capture.bed
chr1 14415 14499 gn|DDX11L1;gn|RP11-34P13.2;ens|ENSG00000223972;ens|ENSG00000227232;vega|OTTHUMG00000000958;vega|OTTHUMG00000000961
chr1 14509 14596 gn|DDX11L1;gn|RP11-34P13.2;ens|ENSG00000223972;ens|ENSG00000227232;vega|OTTHUMG00000000958;vega|OTTHUMG00000000961
chr1 14692 14782 gn|RP11-34P13.2;ens|ENSG00000227232;vega|OTTHUMG00000000958
chr1 14852 14972 gn|RP11-34P13.2;ens|ENSG00000227232;vega|OTTHUMG00000000958
chr1 15032 15128 gn|RP11-34P13.2;ens|ENSG00000227232;vega|OTTHUMG00000000958
chr1 15631 15900 gn|RP11-34P13.2;ens|ENSG00000227232;vega|OTTHUMG00000000958


Could u help me with this problem?

[pravee1216]

has anyone experience this problem?

Has anyone experience the above problem?

[pravee1216]

Another strange behavior of NGSrich

All,

I'm struggling for almost a week in executing NGSrich v0.7.7 on an aligned BAM file with SureSelect All Exon V2 target bed file. The sorted/indexed BAM file (aligned using BWA against hg19 UCSC reference) has the following @SQ

@HD VN:1.0 GO:none SO:coordinate
@SQ SN:chr1 LN:249250621
@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:chr10 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:chr20 LN:63025520
@SQ SN:chr21 LN:48129895
@SQ SN:chr22 LN:51304566
@SQ SN:chrX LN:155270560
@SQ SN:chrY LN:59373566
@SQ SN:chrMT LN:16571
@RG ID:S_4A PL:illumina PU:2 LB:S_4A SM:S_4A
@RG ID:S_4B PL:illumina PU:2 LB:S_4A SM:S_4A


The target capture BED has the following chr#s:

chr1
chr10
chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chr2
chr20
chr21
chr22
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chrX
chrY

and the refGene was downloaded by NGSrich using -u hg19.

GATK DepthOfCoverage was successfully completed on these files, but I have no idea why NGSrich cannot handle this. It started ">>> STEP 2: computing target coverage data" and no response thereafter, keep running for days with ~100% CPU. Either it should return an error then exit, if an exception caught.

Is this an unhandled or never-noticed exception? Any clues would be helpful.

Thanks

Raj

[pravee1216]

Hello All,

I faced a very strange problem with NGSrich (v0.7.6). I was so curious to see the results on my data, hence I invoked NGSrich with the following params (from /bin directory)

java -Xmx4000m NGSrich evaluate -r sample151.bam -u hg19 -t target.bed -o output -a refGene.txt --no-sort -T /tmp -b 14 -m 20

It was so strange that it did not finish, even after running for 2-3 days. Step-1 was completed, but Step-2 (computing target coverage data) was running for such a long time. I noticed the CPU usage which was ~90-95%. Even, I ran without --no-sort and -T and -b/-m options, but there was no change. I don't know whats going wrong here. Input files are the one I had used for all downstream analysis, so no problem with input files.

Please suggest what's wrong here.

Thanks,

Raj.

[@abdallah]

Hi,

@ulz_peter and Lien,
Would you please send me your log files (output and error files), your target file and (ideally) some of the (missed) lines from the read file? We will than try to find the cause.

@brentp,
thank you very much for your suggestions we hope to consider them as soon as time permits.

ps: there is no special reason for this form of invokation of the sort command. Both versions are, to my knowledge, the same.

Regards,
Ali

[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

[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

[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

[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.

[@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

[ulz_peter]

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

[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]

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