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  • #16
    Hi Brian,

    Thanks for the message. Should I leave the file as gzipped or extract it to fast before doing any alignment.

    Thanks,

    Comment


    • #17
      You can leave them gzipped.

      Comment


      • #18
        Hi Brian

        I have been able to run other example parameters efficiently.
        So, I tried to create a index fasta file with the files you mentioned (got if from the JGI website).

        I don't seem to be able to prepare a concatenate index file. I put all the mentioned files in one folder in G: and ran the following command
        java -Xmx1g -ea -cp G:\bbmap\current\align2.BBmap ref=G:\bbmap\lyrata_genome_index

        Folder named lyrata_genome_index contains all three gziped files.
        I got the error message:
        Error: Could not find or laod main class ref=G:\bbmap\lyrata_genome\index

        Again, I tried to prepare the index file for just one file (not including chloroplast and mitochondrial genome).
        I get the same error message.

        Can you please help.

        Also, is there any command to concatenate all these files into one.

        Thanks,

        Comment


        • #19
          Code:
          java -Xmx1g -ea -cp G:\bbmap\current\align2.BBmap ref=G:\bbmap\lyrata_genome_index
          is missing a space; should be:
          Code:
          java -Xmx1g -ea -cp G:\bbmap\current\ align2.BBmap ref=G:\bbmap\lyrata_genome_index
          To concatenate into one file, in Windows:

          Code:
          type file1 file2 > file3
          ...taken from http://stackoverflow.com/questions/6...cat-on-windows

          Comment


          • #20
            Thanks for quick reply.

            Concatenating work perfect.
            I had used spaced in my previous command and again did the same after I was able concatenate all the scaffolds into one.
            But, still getting the error message:
            Error: Could not find or load main class align2.BBmap

            I check and BBmap.java BBmap.class and several other important BBmap. files do exist inside the required folder (current).

            I have been running cmd as administrator in Windows 8.1. HOpefully the problems isn't coming from there.

            Comment


            • #21
              Ah - in this case it's capitalization - should be BBMap, not BBmap. I should have caught that the first time.

              Comment


              • #22
                Thanks Brian.

                Comment


                • #23
                  Hi Brian,

                  I was able to index the genome into one file. Looking at the summary it seems that it is chr1, mitochondrial and chlroplast genome. Thats fine for now. I may be able to add sequences from other chromosomes.

                  For now I tried to map RNAseq reads from one of my sample for the test purpose. I am not worried about splice right now. But, I have been getting errors. I am not sure what the errors mean.
                  I am posting screen shots.

                  Also, to allow splice junction (which I would expect for RNAseq reads) what should I do. I have been reading the readme file. I understand the concept but for some reason whole coding process still doesn't make very good sense to me.

                  I was able to do fastqc quality check on iplant. Is there any parameter in BBMap equivalent to this quality check app.

                  Thanks,
                  Attached Files

                  Comment


                  • #24
                    Once you have the index files ready you use them with path=C:\bbmap\refreads directive (if that path is right). Do not use ref=.

                    Comment


                    • #25
                      Originally posted by everestial View Post
                      Also, to allow splice junction (which I would expect for RNAseq reads) what should I do. I have been reading the readme file. I understand the concept but for some reason whole coding process still doesn't make very good sense to me.
                      Here is a post to help understand minimum options you could use: http://seqanswers.com/forums/showpos...68&postcount=3

                      Read through the thread for several additional pointers that Brian has provided for additional questions.

                      Comment


                      • #26
                        Seems like there is some problem with java. I am not sure.

                        First, I deleted the previous index file and created a new one. The cmd prompt window on the left. There is an error message at the bottom saying
                        Exception in thread.........
                        But, a ref folder is created in G: and there are fasta index files.

                        So, I proceed with mapping one of my RNAseq sample
                        As, the reference had already been set in the previous command I just run align2.BBmap with in=samplename.fasta and out=testF1.fasta
                        But, still getting the same error.
                        Attached Files

                        Comment


                        • #27
                          Originally posted by GenoMax View Post
                          Here is a post to help understand minimum options you could use: http://seqanswers.com/forums/showpos...68&postcount=3

                          Read through the thread for several additional pointers that Brian has provided for additional questions.
                          Well that command for RNAseq reads make a perfect sense. I will follow up with this command and see how my results turn out.

                          Thanks,

                          Comment


                          • #28
                            Originally posted by everestial View Post
                            Seems like there is some problem with java. I am not sure.

                            First, I deleted the previous index file and created a new one. The cmd prompt window on the left. There is an error message at the bottom saying
                            Exception in thread.........
                            But, a ref folder is created in G: and there are fasta index files.

                            So, I proceed with mapping one of my RNAseq sample
                            As, the reference had already been set in the previous command I just run align2.BBmap with in=samplename.fasta and out=testF1.fasta
                            But, still getting the same error.
                            You probably want to create a separate folder when you make the index files that way it would be less confusing to use that path in your ref= part.

                            Can you post the exact commands you are using for:

                            a) to create the index
                            b) to do mapping

                            Screenshots are hard to read.

                            ref= directive only needs to include path up to a top level directory. In that directory there should be a "ref" directory (and additional sub-directories, assuming your index creation worked right).

                            Comment


                            • #29
                              Explanation:
                              I have my F1 reads in F: (due to space concern)
                              I have my bbmap extracted in G:
                              I have my concatenated index reads in G:\bbmap named "indexreads.fq"

                              I run cmd as admin and then move to G: (which I think shouldn't really matter)

                              To prepare the index file I type
                              G:\>java -Xmx1g -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\bbmap\indexreads.fq build=1 genScaffoldInfo=true

                              Message:
                              Executing align2.BBMap [ref=<path to the indexreads.fq>, build=1, genScaffoldInfo=true]

                              BBMap version 34.56
                              Retaining first best site only for ambiguous mappings.
                              No output file.
                              Writing reference.
                              Executing dna.FastaToChromArrays2 [G:\bbmap\indexreads.fq, 1, writeinthread=false, genscaffoldinfo=true, retain, waitforwriting=false, gz=true, maxlen=536670912, writechroms=true, minscaf=1, midpad=300, startpad=8000, stopped=8000, nodisk=false]

                              Set genScaffoldInfo=true
                              Writing chunk 1
                              Set genome to 1

                              Loaded Reference: 4.224 seconds
                              Loading index for chunk 1-1, build 1
                              No index available; generating from reference genome: G:\\ref\index\1\chr1_index_k13_c3_b1.block
                              Indexing threads started for block 0-1
                              Exception in thread "Thread-4" java.lang.OutOfMemoryError: Java heap space at align2.IndexMaker4$BlockMaker$CountThread.run<IndexMaker4.java:280>


                              To map the genome:
                              G:\>java -Xmx1g -e -cp G:\bbmap\current\ align2.BBMap in=F:\F1_extracted\R1_001.fastq out=F:\mapped_test01.sam maxindel=100000 xstag=firststrand intronlen=10 ambig=random

                              The program executes but I ge the same exact message at the bottom starting from Loaded Reference (except for time).

                              Comment


                              • #30
                                Originally posted by everestial View Post
                                G:\>java -Xmx1g -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\bbmap\indexreads.fq build=1 genScaffoldInfo=true

                                Message:
                                Executing align2.BBMap [ref=<path to the indexreads.fq>, build=1, genScaffoldInfo=true]

                                BBMap version 34.56
                                Retaining first best site only for ambiguous mappings.
                                No output file.
                                Writing reference.
                                Executing dna.FastaToChromArrays2 [G:\bbmap\indexreads.fq, 1, writeinthread=false, genscaffoldinfo=true, retain, waitforwriting=false, gz=true, maxlen=536670912, writechroms=true, minscaf=1, midpad=300, startpad=8000, stopped=8000, nodisk=false]

                                Set genScaffoldInfo=true
                                Writing chunk 1
                                Set genome to 1

                                Loaded Reference: 4.224 seconds
                                Loading index for chunk 1-1, build 1
                                No index available; generating from reference genome: G:\\ref\index\1\chr1_index_k13_c3_b1.block
                                Indexing threads started for block 0-1
                                Exception in thread "Thread-4" java.lang.OutOfMemoryError: Java heap space at align2.IndexMaker4$BlockMaker$CountThread.run<IndexMaker4.java:280>
                                You appear to be running out of memory. Increase -Xmx1g to -Xmx4g in the first command. Until this completes without errors do not run the second command.

                                How big is your reference?

                                Comment

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