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  • Need help with Trinity bowtie utility

    I used trinity in iPlant to get a good assembly. However, I have since learned that I should use bowtie and RSEM for mapping and quantifying my transcripts. I successfully downloaded and ran "make" on the Trinity package from Sourceforge and uploaded my Trinity Fasta file and a couple of test fq files to work through the process. However, when I run the following command

    horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110$ util/alignReads.pl --left 0Hr_1_R1.fq --right 0Hr_1_R2.fq --seqType fq --SS_lib_type RF \--target paradormancy_Trinity_contigs.fasta --aligner bowtie2 --retain_intermediate_files -- -p 8

    I get this:

    Error, path to required samtools cannot be found at util/alignReads.pl line 245.
    horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110$

    I ls'd the util file, and didn't see bowtie2. Is there some utility missing from the Trinity package that needs to be added separately,or some other functionality that I need to install? Any advice would be greatly appreciated.

    Dave

  • #2
    Based on the error message, I am guessing the utility that is missing is samtools: http://samtools.sourceforge.net/

    Comment


    • #3
      I downloaded, unzipped and ran make on samtools and place it in my trinity "util" folder, but still get the error:
      Error, path to required samtools cannot be found at util/alignReads.pl line 245.
      horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110$

      Does anyone here have any recent experience downloading and running Trinity all the way through to ID'ing differentially expressed genes from the sourceforge package (http://trinityrnaseq.sourceforge.net/index.html)?

      Comment


      • #4
        Just putting samtools in your util directory may not make it accessible. It is hard to troubleshoot this without getting more information about your system; e.g., does your $PATH include the util directory? did you actually make samtools executable? etc.

        Personally samtools is so useful that I would put it in your bin directory outside of the Trinity directory. In any case if you can do a

        which samtools

        And get output which tells you the path to samtools and/or just be able to type

        samtools

        And get the program to respond to you then Trinity will be able to use samtools. As I said putting samtools somewhere in your normal $PATH is the best bet.

        Comment


        • #5
          Hmm... I am pretty much a novice here (just starting to feel my way around a command line interface) and thus may not be sure I understand what you are saying.
          My current path is: horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$.
          when I ls this I can see a blue folder titled "samtools"
          This folder is the one I unzipped and ran make on.
          I can run "ls samtools" and see all the files and subfolders in that folder.

          When I run
          horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ ./alignReads.pl --left 0Hr_1_R1.fq --right 0Hr_1_R2.fq --seqType fq --SS_lib_type RF \--target paradormancy_Trinity_contigs.fasta --aligner bowtie2 --retain_intermediate_files -- -p 8

          I know it is finding the inputs (because it gave me errors when I didn't have them in the "Util" folder). I also changed the name of the samtools folder to remove the extension so that folder just reads "samtools" but I still get the following error:

          Error, path to required samtools cannot be found at ./alignReads.pl line 245.

          Comment


          • #6
            Did I say to run a 'ls samtools'? While that may help it doesn't show if you actually have the samtools program in your path. Without running a 'which samtools' and just plain old 'samtools' we can not be sure if the program is in your path. And without it being in your path trinity will fail no matter what type of commands you give it.

            My suspicion is that your PATH needs to include:

            /vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools

            Because I suspect that what you have in 'util' is the subdirectory called 'samtools' that contains the actual program 'samtools'.

            Comment


            • #7
              And if you don't know how to set your $PATH ... well then some more reading about how to use the command line interface will be required.

              Comment


              • #8
                @horvathdp Have a look at Part 1 of this excellent guide to UNIX for Biologists: http://korflab.ucdavis.edu/Unix_and_...rl_v3.1.1.html

                Some of the concepts that Rick has referred to would become clear afterwards.
                Last edited by GenoMax; 11-26-2013, 10:37 AM.

                Comment


                • #9
                  Thanks GenoMax, will do here momentarily:
                  @Rick, sorry to frustrate. I did type "which samtools" but nothing happened- even when I cd'd into the samtools directory. see below:


                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ which samtools
                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ samtools
                  bash: samtools: command not found
                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$
                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ cd ./samtools
                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools$ samtools
                  bash: samtools: command not found
                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools$ which samtools
                  horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools$

                  Comment


                  • #10
                    Try this from the directory you are in

                    Code:
                    horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ cd samtools
                    horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ ls -l
                    horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ ./samtools
                    If the compilation worked correctly then an executable file called "samtools" should be in "/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools" directory.

                    What flavor of Unix are you using? If you are using the right terminal type the "samtools" executable may be displayed in green color.
                    Last edited by GenoMax; 11-26-2013, 10:52 AM.

                    Comment


                    • #11
                      horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools$ cd ..

                      #did this to get back up to the utilities directory before running your script.

                      horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ cd samtools
                      horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util/samtools$ ls -l

                      total 4432
                      -rw-r----- 1 horvathdp iplant-everyone 916 Mar 19 2013 AUTHORS
                      -rw-r----- 1 horvathdp iplant-everyone 14804 Mar 19 2013 bam2bcf.c
                      -rw-r----- 1 horvathdp iplant-everyone 1889 Mar 19 2013 bam2bcf.h
                      -rw-r----- 1 horvathdp iplant-everyone 18351 Mar 19 2013 bam2bcf_indel.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 85424 Nov 22 10:41 bam2bcf_indel.o
                      -rw-r--r-- 1 horvathdp iplant-everyone 60456 Nov 22 10:41 bam2bcf.o
                      -rw-r----- 1 horvathdp iplant-everyone 5844 Mar 19 2013 bam2depth.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 23064 Nov 22 10:41 bam2depth.o
                      -rw-r----- 1 horvathdp iplant-everyone 5085 Mar 19 2013 bam_aux.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 49376 Nov 22 10:40 bam_aux.o
                      -rw-r----- 1 horvathdp iplant-everyone 15981 Mar 19 2013 bam.c
                      -rw-r----- 1 horvathdp iplant-everyone 5803 Mar 19 2013 bam_cat.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 20328 Nov 22 10:41 bam_cat.o
                      -rw-r----- 1 horvathdp iplant-everyone 3333 Mar 19 2013 bam_color.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 13888 Nov 22 10:41 bam_color.o
                      -rw-r----- 1 horvathdp iplant-everyone 1064 Mar 19 2013 bam_endian.h
                      -rw-r----- 1 horvathdp iplant-everyone 25785 Mar 19 2013 bam.h
                      -rw-r----- 1 horvathdp iplant-everyone 25788 Mar 19 2013 bam.h~
                      -rw-r----- 1 horvathdp iplant-everyone 16461 Mar 19 2013 bam_import.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 100840 Nov 22 10:41 bam_import.o
                      -rw-r----- 1 horvathdp iplant-everyone 21491 Mar 19 2013 bam_index.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 152960 Nov 22 10:41 bam_index.o
                      -rw-r----- 1 horvathdp iplant-everyone 4976 Mar 19 2013 bam_lpileup.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 42448 Nov 22 10:41 bam_lpileup.o
                      -rw-r----- 1 horvathdp iplant-everyone 4553 Mar 19 2013 bam_mate.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 29312 Nov 22 10:41 bam_mate.o
                      -rw-r----- 1 horvathdp iplant-everyone 12942 Mar 19 2013 bam_md.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 58312 Nov 22 10:41 bam_md.o
                      -rw-r--r-- 1 horvathdp iplant-everyone 89072 Nov 22 10:41 bam.o
                      -rw-r----- 1 horvathdp iplant-everyone 12954 Mar 19 2013 bam_pileup.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 45592 Nov 22 10:41 bam_pileup.o
                      -rw-r----- 1 horvathdp iplant-everyone 22175 Mar 19 2013 bam_plcmd.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 91976 Nov 22 10:41 bam_plcmd.o
                      -rw-r----- 1 horvathdp iplant-everyone 1506 Mar 19 2013 bam_reheader.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 13304 Nov 22 10:41 bam_reheader.o
                      -rw-r----- 1 horvathdp iplant-everyone 5631 Mar 19 2013 bam_rmdup.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 79104 Nov 22 10:41 bam_rmdup.o
                      -rw-r----- 1 horvathdp iplant-everyone 4127 Mar 19 2013 bam_rmdupse.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 60056 Nov 22 10:41 bam_rmdupse.o
                      -rw-r----- 1 horvathdp iplant-everyone 3603 Mar 19 2013 bamshuf.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 55440 Nov 22 10:41 bamshuf.o
                      -rw-r----- 1 horvathdp iplant-everyone 18685 Mar 19 2013 bam_sort.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 142288 Nov 22 10:41 bam_sort.o
                      -rw-r----- 1 horvathdp iplant-everyone 3202 Mar 19 2013 bam_stat.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 18120 Nov 22 10:41 bam_stat.o
                      -rw-r----- 1 horvathdp iplant-everyone 5403 Mar 19 2013 bamtk.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 39184 Nov 22 10:41 bamtk.o
                      -rw-r----- 1 horvathdp iplant-everyone 11391 Mar 19 2013 bam_tview.c
                      -rw-r----- 1 horvathdp iplant-everyone 7879 Mar 19 2013 bam_tview_curses.c
                      -rw-r----- 1 horvathdp iplant-everyone 1880 Mar 19 2013 bam_tview.h
                      -rw-r----- 1 horvathdp iplant-everyone 8406 Mar 19 2013 bam_tview_html.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 61112 Nov 22 10:41 bam_tview.o
                      drwxr-x--- 2 horvathdp iplant-everyone 4096 Nov 22 10:41 bcftools
                      -rw-r----- 1 horvathdp iplant-everyone 3143 Mar 19 2013 bedcov.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 27088 Nov 22 10:41 bedcov.o
                      -rw-r----- 1 horvathdp iplant-everyone 3882 Mar 19 2013 bedidx.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 60128 Nov 22 10:41 bedidx.o
                      -rw-r----- 1 horvathdp iplant-everyone 21127 Mar 19 2013 bgzf.c
                      -rw-r----- 1 horvathdp iplant-everyone 6326 Mar 19 2013 bgzf.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 71288 Nov 22 10:40 bgzf.o
                      -rw-r----- 1 horvathdp iplant-everyone 5903 Mar 19 2013 bgzip.c
                      -rw-r----- 1 horvathdp iplant-everyone 116368 Mar 19 2013 ChangeLog.old
                      -rw-r----- 1 horvathdp iplant-everyone 1085 Mar 19 2013 COPYING
                      -rw-r----- 1 horvathdp iplant-everyone 5667 Mar 19 2013 cut_target.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 31152 Nov 22 10:41 cut_target.o
                      -rw-r----- 1 horvathdp iplant-everyone 3485 Mar 19 2013 errmod.c
                      -rw-r----- 1 horvathdp iplant-everyone 442 Mar 19 2013 errmod.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 32120 Nov 22 10:41 errmod.o
                      drwxr-x--- 2 horvathdp iplant-everyone 4096 Mar 19 2013 examples
                      -rw-r----- 1 horvathdp iplant-everyone 10934 Mar 19 2013 faidx.c
                      -rw-r----- 1 horvathdp iplant-everyone 3188 Mar 19 2013 faidx.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 93096 Nov 22 10:41 faidx.o
                      -rw-r----- 1 horvathdp iplant-everyone 1200 Mar 19 2013 INSTALL
                      -rw-r----- 1 horvathdp iplant-everyone 15230 Mar 19 2013 kaln.c
                      -rw-r----- 1 horvathdp iplant-everyone 2072 Mar 19 2013 kaln.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 39160 Nov 22 10:41 kaln.o
                      -rw-r----- 1 horvathdp iplant-everyone 17815 Mar 19 2013 khash.h
                      -rw-r----- 1 horvathdp iplant-everyone 3472 Mar 19 2013 klist.h
                      -rw-r----- 1 horvathdp iplant-everyone 18332 Mar 19 2013 knetfile.c
                      -rw-r----- 1 horvathdp iplant-everyone 1611 Mar 19 2013 knetfile.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 41800 Nov 22 10:41 knetfile.o
                      -rw-r----- 1 horvathdp iplant-everyone 10675 Mar 19 2013 kprobaln.c
                      -rw-r----- 1 horvathdp iplant-everyone 1568 Mar 19 2013 kprobaln.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 27360 Nov 22 10:41 kprobaln.o
                      -rw-r----- 1 horvathdp iplant-everyone 8811 Mar 19 2013 kseq.h
                      -rw-r----- 1 horvathdp iplant-everyone 10125 Mar 19 2013 ksort.h
                      -rw-r----- 1 horvathdp iplant-everyone 5388 Mar 19 2013 kstring.c
                      -rw-r----- 1 horvathdp iplant-everyone 4459 Mar 19 2013 kstring.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 20048 Nov 22 10:40 kstring.o
                      -rw-r--r-- 1 horvathdp iplant-everyone 1189274 Nov 22 10:41 libbam.a
                      -rw-r----- 1 horvathdp iplant-everyone 3015 Mar 19 2013 Makefile
                      -rw-r----- 1 horvathdp iplant-everyone 2092 Mar 19 2013 Makefile.mingw
                      drwxr-x--- 2 horvathdp iplant-everyone 4096 Mar 19 2013 misc
                      -rw-r----- 1 horvathdp iplant-everyone 28174 Mar 19 2013 NEWS
                      -rw-r----- 1 horvathdp iplant-everyone 17140 Mar 19 2013 padding.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 63256 Nov 22 10:41 padding.o
                      -rw-r----- 1 horvathdp iplant-everyone 20819 Mar 19 2013 phase.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 125840 Nov 22 10:41 phase.o
                      -rw-r----- 1 horvathdp iplant-everyone 24343 Mar 19 2013 razf.c
                      -rw-r----- 1 horvathdp iplant-everyone 4137 Mar 19 2013 razf.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 49784 Nov 22 10:41 razf.o
                      -rw-r----- 1 horvathdp iplant-everyone 4110 Mar 19 2013 razip.c
                      -rw-r----- 1 horvathdp iplant-everyone 6051 Mar 19 2013 sam.c
                      -rw-r----- 1 horvathdp iplant-everyone 2688 Mar 19 2013 sam.h
                      -rw-r----- 1 horvathdp iplant-everyone 21248 Mar 19 2013 sam_header.c
                      -rw-r----- 1 horvathdp iplant-everyone 1597 Mar 19 2013 sam_header.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 77232 Nov 22 10:41 sam_header.o
                      -rw-r--r-- 1 horvathdp iplant-everyone 28544 Nov 22 10:41 sam.o
                      -rw-r----- 1 horvathdp iplant-everyone 2984 Mar 19 2013 sample.c
                      -rw-r----- 1 horvathdp iplant-everyone 396 Mar 19 2013 sample.h
                      -rw-r--r-- 1 horvathdp iplant-everyone 50656 Nov 22 10:41 sample.o
                      -rw-r----- 1 horvathdp iplant-everyone 28252 Mar 19 2013 samtools.1
                      -rw-r----- 1 horvathdp iplant-everyone 14887 Mar 19 2013 sam_view.c
                      -rw-r--r-- 1 horvathdp iplant-everyone 72800 Nov 22 10:41 sam_view.o
                      drwxr-x--- 2 horvathdp iplant-everyone 4096 Mar 19 2013 win32

                      Comment


                      • #12
                        I do not see a "samtools" file (samtools.1 is not it). That means you have not been able to compile samtools correctly.

                        If you are using ubuntu then get "samtools" from the Ubuntu software Center. It would be simpler.

                        Comment


                        • #13
                          Oh!I think I am using Ubuntu. I'll rm the current file (interesting reading so far- thanks!).

                          Comment


                          • #14
                            Originally posted by horvathdp View Post
                            Oh!I think I am using Ubuntu. I'll rm the current file (interesting reading so far- thanks!).
                            DON'T rm samtools.1. It is part of the source code!

                            If you are using Ubuntu then get samtools via software center for now (search for samtools in software center.

                            Comment


                            • #15
                              I didn't rm samtools.1

                              when googling to find the program, I ran across one of your old conversations on what seems to be the exact same issue:
                              Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc


                              in it, there was a Just for kicks, I ran
                              $ sudo apt-get install samtools while in the 'util' directory.

                              Stuff happened :-) :
                              horvathdp@128:/vol-8A5507EB/Trinity/trinityrnaseq_r20131110/util$ sudo apt-get install samtools
                              [sudo] password for horvathdp:
                              Reading package lists... Done
                              Building dependency tree
                              Reading state information... Done
                              The following NEW packages will be installed:
                              samtools
                              0 upgraded, 1 newly installed, 0 to remove and 313 not upgraded.
                              Need to get 494 kB of archives.
                              After this operation, 1,047 kB of additional disk space will be used.
                              Get:1 http://archive.ubuntu.com/ubuntu/ precise/universe samtools amd64 0.1.18-1ubuntu2 [494 kB]
                              Fetched 494 kB in 1s (294 kB/s)
                              Selecting previously unselected package samtools.
                              (Reading database ... 155076 files and directories currently installed.)
                              Unpacking samtools (from .../samtools_0.1.18-1ubuntu2_amd64.deb) ...
                              Processing triggers for man-db ...
                              Setting up samtools (0.1.18-1ubuntu2) ...

                              However, when I ls'd the util directory, I do not see anything new.

                              Comment

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