Question: Problem in installing Trinity.
gravatar for majeedaasim
4 months ago by
United States
majeedaasim20 wrote:

I installed Trinity on my ubunto 16.04. When I test it on example data, Clustering, k-mer building, contig construction and clustering by Inchworm went fine but after that, it shows following error:

bowtie2-build: unrecognized option '--threads'
Bowtie 2 version 2.2.6 by Ben Langmead (,


Usage: bowtie2-build [options]* <reference_in> <bt2_index_base>
    reference_in            comma-separated list of files with ref sequences
    bt2_index_base          write bt2 data to files with this dir/basename
*** Bowtie 2 indexes work only with v2 (not v1).  Likewise for v1 indexes. ***


    -f                      reference files are Fasta (default)
    -c                      reference sequences given on cmd line (as
    --large-index           force generated index to be 'large', even if ref
                            has fewer than 4 billion nucleotides
    -a/--noauto             disable automatic -p/--bmax/--dcv memory-fitting
    -p/--packed             use packed strings internally; slower, less memory
    --bmax <int>            max bucket sz for blockwise suffix-array builder
    --bmaxdivn <int>        max bucket sz as divisor of ref len (default: 4)
    --dcv <int>             diff-cover period for blockwise (default: 1024)
    --nodc                  disable diff-cover (algorithm becomes quadratic)
    -r/--noref              don't build .3/.4 index files
    -3/--justref            just build .3/.4 index files
    -o/--offrate <int>      SA is sampled every 2^<int> BWT chars (default: 5)
    -t/--ftabchars <int>    # of chars consumed in initial lookup (default: 10)
    --seed <int>            seed for random number generator
    -q/--quiet              verbose output (for debugging)
    -h/--help               print detailed description of tool and its options
    --usage                 print this usage message
    --version               print version information and quit

> Error: Encountered internal Bowtie 2 exception (#1)
Command: bowtie2-build --wrapper basic-0 --threads 4 -o 3 /home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_data/test_Trinity_Assembly/trinity_out_dir/chrysalis/inchworm.K25.L25.fa.min100 /home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_data/test_Trinity_Assembly/trinity_out_dir/chrysalis/inchworm.K25.L25.fa.min100 
>  Error, cmd: bowtie2-build --threads 4 -o 3 /home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_data/test_Trinity_Assembly/trinity_out_dir/chrysalis/inchworm.K25.L25.fa.min100 /home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_data/test_Trinity_Assembly/trinity_out_dir/chrysalis/inchworm.K25.L25.fa.min100 1>/dev/null 2>tmp.25936.stderr died with ret 256 at /home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/PerlLib/ line 166. 
          Pipeliner::run(Pipeliner=HASH(0x19ca2c0)) called at ../../Trinity line 1827
main::run_chrysalis("/home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_dat"..., "/home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_dat"..., 200, 500, "RF", "/home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_dat"..., "/home/aasim/Aasim/bin/trinityrnaseq-Trinity-v2.5.0/sample_dat"...) called at ../../Trinity line 1667
    main::run_Trinity() called at ../../Trinity line 1320
    eval {...} called at ../../Trinity line 1319

Trinity run failed. Must investigate error above.

trinity software error • 369 views
ADD COMMENTlink modified 4 months ago by h.mon11k • written 4 months ago by majeedaasim20

Please provide all exact commands that got you to the point of this error.

ADD REPLYlink written 4 months ago by GenoMax42k
gravatar for h.mon
4 months ago by
h.mon11k wrote:

Update your Bowtie2 to a newer version. Checking my local Bowtie2 versions, there is no option --threads for bowtie2-build version 2.2.6, but --threads is available for bowtie2-build version 2.2.9.

edit: do not forget to include more details when asking for help, such as command-line used and software versions used.

ADD COMMENTlink modified 4 months ago • written 4 months ago by h.mon11k

I am using Trinity version 2.5.0 on Ubunto 16.4 I checked my bowtie2 version and it is up to date

bowtie2 --version /usr/bin/bowtie2-align-s version 2.2.6 64-bit Built on lgw01-12 Mon Dec 28 11:09:46 UTC 2015 Compiler: gcc version 5.3.1 20151219 (Ubuntu 5.3.1-4ubuntu1) Options: -O3 -m64 -msse2 -funroll-loops -g3 -Wl,-Bsymbolic-functions -Wl,-z,relro -DPOPCNT_CAPABILITY -DWITH_TBB Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}

ADD REPLYlink written 4 months ago by majeedaasim20

The system-wide version of Bowtie2 is "up to date", but Ubuntu 16.04 is a long-term support release and software versions freeze and start to lag behind. You will have to download, install and add to your PATH a newer version of Bowtie2, luckily, the Bowtie2 devs offer a binary download:

mkdir ~/bin
mv bowtie2- ~/bin/Bowtie2-
export PATH=~/bin/Bowtie2-$PATH

Then you can run Trinity. If you want permanent the change in PATH, add export PATH=~/bin/Bowtie2-$PATH to your .bashrc.

ADD REPLYlink written 4 months ago by h.mon11k
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