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  • #31
    Originally posted by jtjli View Post
    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?

    Comment


    • #32
      Originally posted by rbagnall View Post
      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.

      Comment


      • #33
        Originally posted by PeteH View Post
        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

        Comment


        • #34
          Originally posted by pfrommolt View Post
          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



          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

          Comment


          • #35
            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?

            Originally posted by chariko View Post
            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

            Comment


            • #36
              Originally posted by rbagnall View Post
              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

              Comment


              • #37
                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

                Comment


                • #38
                  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

                  Comment


                  • #39
                    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...
                    Last edited by scalabrin; 07-06-2011, 03:12 AM.

                    Comment


                    • #40
                      Dear community,
                      I just uploaded a new version (0.4.4) with BAM support. Please give it a try.
                      Best, Peter

                      Comment


                      • #41
                        Bug and bam supported in new version

                        Originally posted by scalabrin View Post
                        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
                        Last edited by scalabrin; 07-06-2011, 03:09 AM.

                        Comment


                        • #42
                          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!!!

                          Comment


                          • #43
                            Dear community,

                            I have just set up a mailing list for NGSrich users. Please subscribe at
                            Free, secure and fast downloads from the largest Open Source applications and software directory - SourceForge.net


                            All best,
                            Peter

                            Comment


                            • #44
                              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

                              Comment


                              • #45
                                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

                                Comment

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