Error running QuEST 2.4

[赵信]

Hey,
I'm running QuEST for my bowtie aligned chip-seq reads, and QuEST give me this error which I don't quite understand. Please help me, I really appreciate it!

my command was: /QuEST_2.4/generate_QuEST_parameters.pl -bowtie_align_ChIP ./SRR016812_sep3_all_out -bowtie_align_RX_noIP ./wt_bowtie -rp /home/chromosomes/TAIR9/ -ap ./QuEST_analysis -ChIP_name SEP3_work1

and QuEST generates this output and error:
QuEST ChIP-Seq analysis pipeline version 2.404
Last modified on 08-21-2009

For questions, suggestions, bugs please contact
Anton Valouev val...@stanford.edu

Please cite the following publication when using QuEST:

Valouev A, Johnson DS, Sundquist A, Medina C, Anton E, Batzoglou S,
Myers RM, Sidow A (2008). Genome-wide analysis of transcription
factor binding sites based on ChIP-Seq data.
Nat Methods 5, 829-834 (2008)


=====================

parameters:

ChIP_name:                     SEP3_work1
ChIP_align_file:               ./SRR016812_sep3_all_out
ChIP_file_type:                bowtie_align

RX_noIP_align_file:            ./wt_bowtie
RX_noIP_file_type:             bowtie_align

reference path:                /home/xin/chromosomes/TAIR9/
genome_table:                  ** missing **

analysis_path:                 /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis

silent:                        off
advanced:                      off

=====================

creating necessary directories.

- - - - - - - - - - - - - - - -

analysis directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis:   created
logs directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/logs: created
module outputs directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/module_outputs: created
parameters directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/parameters: created
calls directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/calls: created
data directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/data: created
scores directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/scores: created
tracks directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks: created
bin_align directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/bin_align: created
bin_align ChIP directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/bin_align/ChIP: created
bin_align background directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/bin_align/background: created
bin_align RX_noIP directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/bin_align/RX_noIP: created
bin_align pseudo_ChIP directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/bin_align/pseudo_ChIP: created
bed graph tracks directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/bed_graph: created
bed graph tracks by chromosome directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/bed_graph/by_chr: created
bed graph tracks by chromosome ChIP directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/bed_graph/by_chr/ChIP: created
data bed tracks directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/data_bed_files: created
data bed tracks by chromosome directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/data_bed_files/by_chr: created
ChIP bed tracks by chromosome directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/data_bed_files/by_chr/ChIP: created
RX_noIP bed tracks by chromosome directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/data_bed_files/by_chr/RX_noIP: created
wig tracks directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/wig_profiles: created
wig tracks by chromosome directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/wig_profiles/by_chr: created
wig tracks by chromosome directory for normalized ChIP profiles: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/wig_profiles/by_chr/ChIP_normalized: created
wig tracks by chromosome directory for unnormalized ChIP profiles: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/wig_profiles/by_chr/ChIP_unnormalized: created
wig tracks by chromosome directory for normalized backround profiles: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/wig_profiles/by_chr/background_normalized: created
wig tracks by chromosome directory for unnormalized background profiles: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/tracks/wig_profiles/by_chr/background_unnormalized: created
ChIP scores directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/scores/ChIP: created
background scores directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/scores/background: created
pseudo_ChIP scores directory: /media/Elements/xin/chip_seq/SEP3 work1/QuEST_analysis/scores/pseudo_ChIP: created

- - - - - - - - - - - - - - - -


The following fasta files were found in your reference path:

chr3.fa
chrM.fa
chr1.fa
chrC.fa
chr2.fa
chr5.fa
chr4.fa

Evaluating the size of the genome. This may take several minutes.

Found the following contigs:

Chr3 CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2009-02-02: 23459830 bps
mitochondria CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2005-06-03: 366924 bps
Chr1 CHROMOSOME dumped from ADB: Jun/20/09 14:53; last updated: 2009-02-02: 30427671 bps
chloroplast CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2005-06-03: 154478 bps
Chr2 CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2009-02-02: 19698289 bps
Chr5 CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2009-02-02: 26975502 bps
Chr4 CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2009-02-02: 18585056 bps

Your genome looks fine and does not appear to contain any
duplicated chromosome entries.

Estimated size of the genome is 119667.8 Kb ( 0.12 Gbps ).

Converting ChIP reads file into the QuEST format.
If the counter below stays at zero, the program can't
match the ChIP alignments to the reference genome that you provided.
In this case check that the sequences you have provided match the formats
accepted by QuEST (see README.txt) and that names of contigs in the alignment
files are *exactly* the same as in your genome or genome table.

Warning:
couldn't parse the parameter: work1/QuEST_analysis/data/ChIP_uncollapsed.QuEST
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/parameters/genome_table
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/data/ChIP_read_num_uncollapsed.txt
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/module_outputs/align_2_QuEST_ChIP.report.txt
skipping
align_file:         ./SRR016812_sep3_all_out
output_file:        /media/Elements/xin/chip_seq/SEP3
genome_table:       /media/Elements/xin/chip_seq/SEP3
alignment_type:     bowtie
report_file:        /media/Elements/xin/chip_seq/SEP3
error_report_file:  /media/Elements/xin/chip_seq/SEP3

aligments: 1.55 M, matched : 1.55 M, not in gt: 0.000 M, offending: 0.000 M

Lines read: 1554441
Alignments matched: 1554441
Offending lines: 0

Checking for duplicate hits...
[ This screens for contiguity of bowtie alignment order ]
Your bowtie alignment file seems to be ok.


readline() on closed filehandle ChIP_uncollapsed_read_num_file at
        /home/xin/software/QuEST_2.4/generate_QuEST_parameters.pl line 887 (#1)
    (W closed) The filehandle you're reading from got itself closed sometime
    before now.  Check your control flow.
   
Use of uninitialized value $ChIP_uncollapsed_tags in scalar chomp at
        /home/xin/software/QuEST_2.4/generate_QuEST_parameters.pl line 891 (#2)
    (W uninitialized) An undefined value was used as if it were already
    defined.  It was interpreted as a "" or a 0, but maybe it was a mistake.
    To suppress this warning assign a defined value to your variables.
   
    To help you figure out what was undefined, perl will try to tell you the
    name of the variable (if any) that was undefined. In some cases it cannot
    do this, so it also tells you what operation you used the undefined value
    in.  Note, however, that perl optimizes your program and the operation
    displayed in the warning may not necessarily appear literally in your
    program.  For example, "that $foo" is usually optimized into "that "
    . $foo, and the warning will refer to the concatenation (.) operator,
    even though there is no . in your program.
   
Collapsing ChIP data...
Warning:
couldn't parse the parameter: work1/QuEST_analysis/data/ChIP_uncollapsed.QuEST
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/bin_align/ChIP
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/parameters/genome_table
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/data/ChIP.QuEST
skipping
Warning:
couldn't parse the parameter: work1/QuEST_analysis/data/ChIP_read_num.txt
skipping

Specified parameters:
executable      /home/xin/software/QuEST_2.4/collapse_reads
align_file      /media/Elements/xin/chip_seq/SEP3

output_path     /media/Elements/xin/chip_seq/SEP3

genome_table    /media/Elements/xin/chip_seq/SEP3

QuEST_collapsed_file    /media/Elements/xin/chip_seq/SEP3

collapse_reads  true
collapse_window 100
count_threshold 2
stack_p_value_threshold 0.001
percent_positions_hit_threshold 30
report_file     /media/Elements/xin/chip_seq/SEP3

new_stack_size  1
read 0.00 M reads

sorting hits
done!

readline() on closed filehandle ChIP_read_num_file at
        /home/xin/software/QuEST_2.4/generate_QuEST_parameters.pl line 1099 (#1)
Use of uninitialized value $ChIP_tags in scalar chomp at
        /home/xin/software/QuEST_2.4/generate_QuEST_parameters.pl line 1104 (#2)
Use of uninitialized value $ChIP_tags in concatenation (.) or string at
        /home/xin/software/QuEST_2.4/generate_QuEST_parameters.pl line 1105 (#2)
After collapsing, there are  ChIP read alignments
Use of uninitialized value $ChIP_tags in numeric le (<=) at
        /home/xin/software/QuEST_2.4/generate_QuEST_parameters.pl line 1107 (#2)
No ChIP reads were found in your data. Please check your alignment files.



Thank you so much!
Xin

[Anton]

Hi Xin,

I've seen this error before, but I didn't have enough info from the user to tell what was causing it.

I suspect the bowtie alignments might be the problem. 

Can you please try saving the alignments as SAM files and then running QuEST again?

Best, Anton