Detonate - Trinity De Novo Assembly Evaluator Error - invalid number of arguments when trying to execute the rsem-eval-calculate score command

Question

[sierraallinone]

I have paired-end Illumina Files named

Mantle_TT_7_2_TGACCAqtrim.fq Mantle_TT_7_1_TGACCAqtrim.fq

The 7_2 file being the forward and 7_1 being the reverse.

The name of the Trinity Output Assembly file is TrinityMantleTT.fasta

the paths to these files are:

~/Desktop/Mantle_TT_7_2_TGACCAqtrim.fq
~/Desktop/Mantle_TT_7_1_TGACCAqtrim.fq
~/Desktop/TrinityMantleTT.fasta

I'm trying to run this with the following command:

perl rsem-eval-calculate-score --paired-end ~/Desktop/Mantle_TT_7_2_TGACCAqtrim.fq \
    ~/Desktop/Mantle_TT_7_1_TGACCAqtrim.fq \
    ~/Desktop/TrinityMantleTT.fasta Mantle_TT_rsem_eval

But I keep getting an error saying invalid number of argumentss

Am I listing my paired-end files incorrectly? Am I missing something?

Please help

[sierraallinone]

SYNOPSIS

 rsem-eval-calculate-score [options] upstream_read_file(s) assembly_fasta_file sample_name L
 rsem-eval-calculate-score [options] --paired-end upstream_read_file(s) downstream_read_file(s) assembly_fasta_file sample_name L
 rsem-eval-calculate-score [options] --sam/--bam [--paired-end] input assembly_fasta_file sample_name L

ARGUMENTS

upstream_read_files(s)
    Comma-separated list of files containing single-end reads or
    upstream reads for paired-end data. By default, these files are
    assumed to be in FASTQ format. If the --no-qualities option is
    specified, then FASTA format is expected.

downstream_read_file(s)
    Comma-separated list of files containing downstream reads which are
    paired with the upstream reads. By default, these files are assumed
    to be in FASTQ format. If the --no-qualities option is specified,
    then FASTA format is expected.

input
    SAM/BAM formatted input file. If "-" is specified for the filename,
    SAM/BAM input is instead assumed to come from standard input.
    RSEM-EVAL requires all alignments of the same read group together.
    For paired-end reads, RSEM-EVAL also requires the two mates of any
    alignment be adjacent. See Description section for how to make input
    file obey RSEM-EVAL's requirements.

assembly_fasta_file
    A multi-FASTA file contains the assembly used for calculating
    RSEM-EVAL score.

sample_name
    The name of the sample analyzed. All output files are prefixed by
    this name (e.g., sample_name.isoforms.results).

L   For single-end data, L represents the average read length. For
    paired-end data, L represents the average fragment length. It should
    be a positive integer (real value will be rounded to the nearest
    integer).

BASIC OPTIONS

--overlap-size <int>
    The minimum overlap size required to join two reads together.
    (Default: 0)

--transcript-length-parameters <file>
    Read the true transcript length distribution's mean and standard
    deviation from <file>. This option is mutually exclusive with
    '--transcript-length-mean' and '--transcript-length-sd'. (Default:
    off)

--transcript-length-mean <double>
    The mean of true transcript length distribution. This option is used
    together with '--transcript-length-sd' and mutually exclusive with
    '--estimate-transcript-length-distribution'. (Default: learned from
    human Ensembl annotation and hg20 genome)

--transcript-length-sd <double>
    The standard deviation of true transcript length distribution. This
    option is used together with '--transcript-length-mean' and mutually
    exclusive with '--estimate-transcript-length-distribution'.
    (Default: learned from human Ensembl annotation and hg20 genome)

--paired-end
    Input reads are paired-end reads. (Default: off)

--no-qualities
    Input reads do not contain quality scores. (Default: off)

--strand-specific
    The RNA-Seq protocol used to generate the reads is strand specific,
    i.e., all (upstream) reads are derived from the forward strand. This
    option is equivalent to --forward-prob=1.0. With this option set, if
    RSEM-EVAL runs the Bowtie/Bowtie 2 aligner, the '--norc'
    Bowtie/Bowtie 2 option will be used, which disables alignment to the
    reverse strand of transcripts. (Default: off)

--bowtie2
    Use Bowtie 2 instead of Bowtie to align reads. Since currently
    RSEM-EVAL does not handle indel, local and discordant alignments,
    the Bowtie2 parameters are set in a way to avoid those alignments.
    In particular, we use options '--sensitive --dpad 0 --gbar 99999999
    --mp 1,1 --np 1 --score-min L,0,-0.1' by default. "-0.1", the last
    parameter of '--score-min' is the negative value of the maximum
    mismatch rate allowed. This rate can be set by option
    '--bowtie2-mismatch-rate'. If reads are paired-end, we additionally
    use options '--no-mixed' and '--no-discordant'. (Default: off)

-p/--num-threads <int>
    Number of threads to use. Both Bowtie/Bowtie2, expression estimation
    and 'samtools sort' will use this many threads. (Default: 1)

[sierraallinone]

Here is the example they give:

EXAMPLES

We want to compute the RSEM-EVAL score for a contig assembly,
'assembly1.fa'. Our data are 76bp single-end reads contained in
'/data/reads.fq'. The related species is human and
'human_transcripts.fa' contains all human transcripts. We use 8 threads
and do not generate any BAM files. In addition, we set the overlap size
w as 0 and 'sample_name' as 'assembly1_rsem_eval'.

First, we need to estimate the true transcript length distribution using
'human_transcripts.fa':

 rsem-eval-estimate-transcript-length-distribution human_transcripts.fa human.txt

 Now, we can calculate RSEM-EVAL score:

 rsem-eval-calculate-score -p 8 \
                           --transcript-length-parameters human.txt \
                           /data/reads.fq \
                           assembly1.fa \
                           assembly1_rsem_eval \
                           76

 The RSEM-EVAL score can be found in 'assembly1_rsem_eval.score' and the
contig impact scores can be found in
'assembly1_rsem_eval.score.isoforms.results'.

[h.mon]

Probably not the cause of the error, but programs in general expect f1.fq f2.fq, not f2.fq f1.fq:

--paired-end ~/Desktop/Mantle_TT_7_1_TGACCAqtrim.fq ~/Desktop/Mantle_TT_7_2_TGACCAqtrim.fq