0 peaks found using QuEST

[nultontj]

I'm using QuEST to analyze our labs chIP-seq data and comparing it with the analysis done by our informatician who is using a different approach.  However, when I run the analysis, this is what I get...

Found the following contigs:

gi|125716887|ref|NC_009009.1| Streptococcus sanguinis SK36 chromosome, complete genome: 2388435 bps

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

Estimated size of the genome is 2388.4 Kb ( 0.00 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.

align_file:         not_pur_959.sam
output_file:        /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP_uncollapsed.QuEST
genome_table:       /home/nultontj/july_chIP_959/QuEST_analysis/parameters/genome_table
alignment_type:     bowtie
report_file:        /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP_read_num_uncollapsed.txt
error_report_file:  /home/nultontj/july_chIP_959/QuEST_analysis/module_outputs/align_2_QuEST_ChIP.report.txt

Bad contig size 0 in gi|125716887|ref|NC_009009.1| Streptococcus sanguinis SK36 chromosome, complete genome 2388435. Skipping.
aligments: 81.81 M, matched : 0.00 M, not in gt: 0.000 M, offending: 81.810 M

Lines read: 81813685
Alignments matched: 0
Offending lines: 81813685

Checking for duplicate hits...
[ This screens for contiguity of bowtie alignment order ]
Seems like there are no alignments to check. Are you sure your bowtie file is ok?


Collapsing ChIP data...

Specified parameters:
executable      /home/nultontj/july_chIP_959/QuEST_2.4/collapse_reads
align_file      /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP_uncollapsed.QuEST
output_path     /home/nultontj/july_chIP_959/QuEST_analysis/bin_align/ChIP
genome_table    /home/nultontj/july_chIP_959/QuEST_analysis/parameters/genome_table
QuEST_collapsed_file    /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP.QuEST
collapse_reads  true
collapse_window 100
count_threshold 2
stack_p_value_threshold 0.001
percent_positions_hit_threshold 30
report_file     /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP_read_num.txt
new_stack_size  1
read 0.00 M reads

sorting hits

-------------------------
contig: gi|125716887|ref|NC_009009.1|

+ reads: 0
- reads: 0

+ reads after collapsing: 0
- reads after collapsing: 0

stacks collapsed: 0
reads in collapsed stacks: 0
done!

After collapsing, there are 0 ChIP read alignments
No ChIP reads were found in your data. Please check your alignment files.

I've mapped the contigs with bowtie2, not specifying the -z option.  This has resulted in the .sam

files  WT (background and control) and FLAGged gene 959. 

The mapped files do contain identical chromosome name as the .fa file used instead of the genome table.

What is in grey is what I think may be causing the problem, either that or somehow the bowtie files aren't right.

I would really appreciate any help you may be able to give me.

Regards,
Tara

[Anton]

Hi Tara,

contig names are not allowed to have spaces. Try chopping down the names and re-running alignment and QuEST.

[Tara J Nulton]

Thanks!  Will do, Tara

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--
Tara Nulton | The Philips Institute | Virginia Commonwealth University
Wood Memorial Building, Rm 401
521 N 11th Street
Richmond, VA 23298

[Tara J Nulton]

Here is a sample of my raw reads files:  As you can see below, this is the space I think you are referring too.

@HWI-ST425:169:D11BJACXX:1:1101:1944:1975 1:N:0:CCGTCC
NTTGCAGCCCGCTGCCGTTCTCCGCCAGACAATTCTTTCGGATACTGCTTGAGAGTTCCTTTAAGATCCAGCAACTCAATCAGACTGTCTAGATCCGTCCTTTCCTTTTT
+
#4=BDDBDHDHDDHFGGGIGGIBHGIEHGEHIAHEIGGCEB1;@3=@AGHG;E;A7;7?C@>A:;A>;@C@ACCB:A55:(5@A@CCCCCC:@A>><?<&8>C:+4>C@C
@HWI-ST425:169:D11BJACXX:1:1101:3365:1965 1:N:0:CCGTCC
NAAATTAAGAGAAGATATCCGTAACATTGCGATTATCGCCCACGTTGACCACGGGAAAACAACCCTTGTTGACGAATTATTGAAACAGTCTGAAACTCTTGATGCTCGTA
+
#1BDFFFFHHHHHJJJJJJJJHIJJJJJJJJJJJJJJJIJJJIJIJIJJJJJJJJHHHFFFFDDDEDDDDDDDDD@BDDEFDDDDDDCACDDDDDDCDDDDDDDDDDDBD
@HWI-ST425:169:D11BJACXX:1:1101:4057:1980 1:N:0:CCGTCC
NAAAGCATGCTCCGTCTCAATAAATGAACAGGCCGCCAAATGTGTCCATAAATTCATCTGCTACCTCTACAAAAAATAAGCGAAAAACGCTTATTCTTCTATCAACTGGA

I got rid of that space and created this file:

@HWI-ST425:169:D11BJACXX:1:1101:1944:19751:N:0:CCGTCC
NTTGCAGCCCGCTGCCGTTCTCCGCCAGACAATTCTTTCGGATACTGCTTGAGAGTTCCTTTAAGATCCAGCAACTCAATCAGACTGTCTAGATCCGTCCTTTCCTTTTT
+
#4=BDDBDHDHDDHFGGGIGGIBHGIEHGEHIAHEIGGCEB1;@3=@AGHG;E;A7;7?C@>A:;A>;@C@ACCB:A55:(5@A@CCCCCC:@A>><?<&8>C:+4>C@C
@HWI-ST425:169:D11BJACXX:1:1101:3365:19651:N:0:CCGTCC
NAAATTAAGAGAAGATATCCGTAACATTGCGATTATCGCCCACGTTGACCACGGGAAAACAACCCTTGTTGACGAATTATTGAAACAGTCTGAAACTCTTGATGCTCGTA
+
#1BDFFFFHHHHHJJJJJJJJHIJJJJJJJJJJJJJJJIJJJIJIJIJJJJJJJJHHHFFFFDDDEDDDDDDDDD@BDDEFDDDDDDCACDDDDDDCDDDDDDDDDDDBD
@HWI-ST425:169:D11BJACXX:1:1101:4057:19801:N:0:CCGTCC
NAAAGCATGCTCCGTCTCAATAAATGAACAGGCCGCCAAATGTGTCCATAAATTCATCTGCTACCTCTACAAAAAATAAGCGAAAAACGCTTATTCTTCTATCAACTGGA

I ran that file through bowtie2 using this command:

bowtie2 -x sk36_index -U nspace_pur_sk36.fastq 

-S bowtied_pur_sk36.sam

This outputted me this file:

@HD     VN:1.0  SO:unsorted
@SQ     SN:gi|125716887|ref|NC_009009.1|        LN:2388435
@PG     ID:bowtie2      PN:bowtie2      VN:2.1.0
HWI-ST425:169:D11BJACXX:1:1101:1398:19871:N:0:CGATGT    0       gi|125716887|ref|NC_009009.1|   132353  42      110M    *       0       0       TGGATGGGCTGCCTGTGCGGACCATGTCTATTCTCTTTAATATCGGGACGGGTGTTGCGATTGCCCTCATGATTCCAGCTGCGGGAGCCTTGCTAGTTTCGGCCATTATG       @@@DD?D=CFFADGAFHIIIGIIHBGGFHAC>FGHGHGGHE>BFE(<AAAGE'5,;=CB>A??>CCCBACCACCC::@C@C#############################       AS:i:-4 XN:i:0  XM:i:2  XO:i:0  XG:i:0  NM:i:2  MD:Z:45T55A8    YT:Z:UU
HWI-ST425:169:D11BJACXX:1:1101:1591:19761:N:0:CGATGT    0       gi|125716887|ref|NC_009009.1|   1347014 42      110M    *       0       0       NTTTTAGATTTCCAAAAACCATACTGCGCATAATTCATCAACTCTTTGCCAGTATGTTCTCCCGACATGTAACTATCTGCGGCTTCTTTTGGAATTTCTCTAATCACTTC       #1=DFDFEHHHHFIIJJJIJJJJJIJHHGIJIIIJDGIIJJJJJJJJIIJIIIIIJHIIJJJJJJJHHHHFFFFFBEEEEDDDDDCCDDD@>:>ACD@D@C:@C::>@C>       AS:i:-1 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:0T109      YT:Z:UU
HWI-ST425:169:D11BJACXX:1:1101:2329:19921:N:0:CGATGT    0       gi|125716887|ref|NC_009009.1|   1763437 42      110M    *       0       0       GAGGATTCACCGTATACAACGACGGTATTGCCCACTACAACAGCTCACTGATTACTTACCTGGTCAGCTTTGGGGTCTTGGCTTTCGGGGTCAACTTTAACCTCTATTAT       CCCFFFFFHHHHHJJJJJJJJJJJJGHJJJJJJIJJJJJJJJJJJJGIIJJJJIJJJJJHHHHHFFFFFFECEDDDBCDDDDDDDC@D@B>@ACDDDDCCDDDDDACDCD       AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:110        YT:Z:UU
HWI-ST425:169:D11BJACXX:1:1101:5541:19671:N:0:CGATGT    0       gi|125716887|ref|NC_009009.1|   133849  42      110M    *       0       0       NACATCACAGACAAGACTATGGAATTTGTGGTAGATGGAAAATCCAAGAAATATACTTATAAATACGTTGGTAAGCATACTCTGACTTACTCTAAGGGAAATCGTGGAGT       #1BDFFFFHHHHHJJIJJJJJJJJJJJJFHICGHIGIIJJJJIIIIIIIJJJJIJJJJJJJJJJJJJHIJJIIJHCHHHHFFFFFFEEEEEEEDDDDDDDDDDDDDD@DC       AS:i:-1 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:0A109      YT:Z:UU
HWI-ST425:169:D11BJACXX:1:1101:6231:19691:N:0:CGATGT    0       gi|125716887|ref|NC_009009.1|   250675  27      110M    *       0       0       NTCACCTCTATTTTGTGAATGATGCAGGTCAGTTGCAGGGTGCAGGCTCAACTAAGACAGTGGTTACTCGCGCCCAGCCACAGGGAAAAATCACCATTCAGAACAACAAT       #1:?DD;DBFDHFEGAEEHGEHGDEEHGIHICGIEFCHIICGEHGGHCHAGIEHIIEGEH=@FFGDH@@@B85AC9?ABBCB<AB?BBCBACC4<:::CCACCCCCABB#       AS:i:-1 XS:i:-59        XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:0T109      YT:Z:UU
HWI-ST425:169:D11BJACXX:1:1101:6550:19721:N:0:CGATGT    0       gi|125716887|ref|NC_009009.1|   908018  42      110M    *       0       0       NTGGCACTCCAGTAACTGCGACATACAGTCCAGAGTTCGCCAAGGTAATTCCAACTGGTAAAGATGCTACATCTACAAATATCAAGGGCCATGTTCAAACTGGCAAACCT       #1=DDDFFDHHFFEGEHJJIGIGGIJIJGHIJFGHIIHGJJJJJIBFHGGG>HIIIEGCEHIIJGHGHHHFFFFFBCEEEEECCDC@@BD?CCCDECCCDDDCDCDDBBC       AS:i:-1 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:0A109      YT:Z:UU
HWI-ST425:169:D11BJACXX:1:1101:6938:19881:N:0:CGATGT    16      gi|125716887|ref|NC_009009.1|   901617  42      110M    *       0       0       AGTGACTGCGACCTACAGTCCAGAGTTCACCAAGGTAACTCCGACAGGTACTGGTACCAAGACAGAAGGTCTGCAAGGTCAGGTTCAAAAAGGTCAAGTGACTTTTACGC       @CA?8>9@?CDDCCAACDDC@ACDDCCA>CEFEB;DB;HHIHGCJJIHIGJIIIGIJIJJIGIEIGHFBBEJIGIIIIGIJIGIIHIJJJIIJIJJGHHDCHFFFFD@@@       AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:110        YT:Z:UU

Which I then used the WT and FLAGGED 959 file .sam formats to run QuEST using:

QuEST_2.4/generate_QuEST_parameters.pl

-bowtie_align_ChIP not_pur_959.sam -bowtie_align_RX_noIP not_pur_sk36.sam

-gt sk36_index.gt -ap ./QuEST_analysis -ChIP_name 959

Still it is giving me no peaks.  Its not breaking, its just continuing to behave in the same manor:



 
chr 2388435

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

Estimated size of the genome is 2388.4 Kb ( 0.00 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.

align_file:         nspace_not_pur_959.fastq

output_file:        /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP_uncollapsed.QuEST
genome_table:       /home/nultontj/july_chIP_959/QuEST_analysis/parameters/genome_table
alignment_type:     bowtie
report_file:        /home/nultontj/july_chIP_959/QuEST_analysis/data/ChIP_read_num_uncollapsed.txt
error_report_file:  /home/nultontj/july_chIP_959/QuEST_analysis/module_outputs/align_2_QuEST_ChIP.report.txt

aligments: 3.21 M, matched : 0.00 M, not in gt: 0.000 M, offending: 3.210 M

I continue to appreciate any help you can give me....

Thanks,

Tara

[Tara J Nulton]

Any suggestions where this is all going wrong?  It's not breaking, just not finding anything and this is not the result obtained from another tool.

Please advise :)

Thanks,
Tara

[Anton]

Hi Tara, sorry for the late response.

It seems like it still doesn't understand your alignments, because all your alignments are "offending".

Bowtie format is no longer supported. Probably best for you would be to edit your original genome fasta file to get rid of spaces in the config names. Then you can align the data using BWA and input the SAM files into QuEST. Then it should work.

Best, Anton

[Tara J Nulton]

Thanks for your response Anton.  I'll realign with BWA and try again!

[Tara J Nulton]

Hello!

I've taken the data and realigned using BWA. 

For BWA I indexed the reference genome, using the index command, found the SA coords using the aln command and generated the sam format using the samse command.

Those SAM files I then fed into QuEST using the same command as before, however I feel like my flags for for QuEST_parameters.pl are wrong because they are for bowtie and QuEST is not recognizing this format.  I could not see in the README file what flag should be used with BWA.  Not solexa, or eland, or bowtie.....

So, I resorted to using the bowtie flags even though these are not bowtie alignments.  Which flag should I be using for the BWA output?

Below is my command:

 QuEST_2.4/generate_QuEST_parameters.pl -bowtie_align_ChIP not_pur959.sam -bowtie_align_RX_noIP not_pursk36.sam -gt sk36_index.gt -ap ./tuesdayquest

Below is a sample...

parameters:

ChIP_name:                     ChIP
ChIP_align_file:               not_pur959.sam
ChIP_file_type:                bowtie_align

RX_noIP_align_file:            not_pursk36.sam
RX_noIP_file_type:             bowtie_align

reference path:                ** missing **
genome_table:                  sk36_index.gt

analysis_path:                 /home/nultontj/july_chIP_959/tuesdayquest

silent:                        off
advanced:                      off

Part of the error file found in  module_outputs/align_2_QuEST_ChIP.report.txt

Line: 1 string: @SQ     SN:gi|125716887|ref|NC_009009.1|        LN:2388435
Too few fields

Line: 2 string: @PG     ID:bwa  PN:bwa  VN:0.6.1-r104
Too few fields

Alignment: HWI-ST425:169:D11BJACXX:1:1101:1944:1975     16      gi|125716887|ref|NC_009009.1|   935228  37      110M    *       0       0       AAAAAGGAAAGGACGGATCTAGACAGTCTGATTGAGTTGCTGGATCTTAAAGGAACTCTCAAGCAGTATCCGAAAGAATTGTCTGGCGGAGAACGGCAGCGGGCTGCAAN  C@C>4+:C>8&<?<>>A@:CCCCCC@A@5(:55A:BCCA@C@;>A;:A>@C?7;7A;E;GHGA@=3@;1BECGGIEHAIHEGHEIGHBIGGIGGGFHDDHDHDBDDB=4#  XT:A:U  NM:i:1  X0:i:1  X1:i:0  XM:i:1  XO:i:0  XG:i:0  MD:Z:109T0
Expected +/- in the 2nd field, but found: 16

Alignment: HWI-ST425:169:D11BJACXX:1:1101:3365:1965     0       gi|125716887|ref|NC_009009.1|   632822  37      110M    *       0       0       NAAATTAAGAGAAGATATCCGTAACATTGCGATTATCGCCCACGTTGACCACGGGAAAACAACCCTTGTTGACGAATTATTGAAACAGTCTGAAACTCTTGATGCTCGTA  #1BDFFFFHHHHHJJJJJJJJHIJJJJJJJJJJJJJJJIJJJIJIJIJJJJJJJJHHHFFFFDDDEDDDDDDDDD@BDDEFDDDDDDCACDDDDDDCDDDDDDDDDDDBD  XT:A:U  NM:i:1  X0:i:1  X1:i:0  XM:i:1  XO:i:0  XG:i:0  MD:Z:0A109
Expected +/- in the 2nd field, but found: 0

Please advise and thank you for continued patience!

Tara

[Anton]

Hi Tara,

if you look at the output of simply running generate_QuEST_parameters.pl

you will see -sam_align_ChIP and -sam_align_RX_noIP are the two flags to use with SAM files.

Best, Anton

[Tara J Nulton]

Anton,

I see no such flag.  Below is the output of running generate_QuEST_parameters.pl


   

QuEST parametrization script

    -----------------------
    mandatory arguments:
    -----------------------

    for the mapped ChIP data use one of the following:

    -QuEST_align_ChIP <QuEST_align_file>
    -solexa_align_ChIP <solexa_ChIP_align_file>
    -eland_align_ChIP <eland_file>
    -eland_extended_ChIP <eland_extended_file>
    -bowtie_align_ChIP <bowtie_file>
    -maq_align_ChIP <maq_file>

    - - - - - - - - - - - -

    for the mapped background data (RX_noIP, input, total chromatin, control) use one of the following:

    -QuEST_align_RX_noIP <QuEST_align_RX_noIP_file>
    -solexa_align_RX_noIP  <solexa_RX_noIP_align_file>
    -eland_align_RX_noIP <eland_file>
    -eland_extended_RX_noIP <eland_extended_file>
    -bowtie_align_RX_noIP <bowtie_file>
    -maq_align_RX_noIP <maq_file>

I try to use the same flags you specified, and the result is:


/home/nultontj/july_chIP_959/QuEST_2.4/generate_QuEST_parameters.pl -sam_align_ChIP not_pur_959.sam -sam_align_RX_noIP not_pur_sk36.sam -gt sk36_index.gt -ap ./QuEST_analysis -ChIP_name 959

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)

Warning: unknown parameter: -sam_align_ChIP

Regards,
Tara

[Tara J Nulton]

In mining the help, I came across this

Thanks Anton, that did it!  Just a heads up the QuEST 2.4 download on
the main page (on the right) and the tutorial page
http://www.stanford.edu/~valouev/QuEST/QuEST.html are different files
(94 and 96 kb respectively).  The main page download can't run sam
files.  All the best!
Jim

perhaps this is the source of my error, and one I will explore further!

[Tara J Nulton]

Anton,

I believe this is the root of the problem, however I find that access to that download is forbidden.

Please advise and thank you for your continued patience.

Tara

[Anton]

Hi Tara, looks like you are using an old version of QuEST. You can grab a 2.4 over here:

http://www-hsc.usc.edu/~valouev/QuEST/QuEST.html

[Tara J Nulton]

Dear Anton,

I had to put this project down for awhile, but its back on my front burner.

Good news, as the newer version of QuEST is behaving better.  The bad news is I'm still getting no alignments.

For indexed the ref genome using BWA,  index command, found the SA coords using the aln command and generated the sam format using the samse command.

Those SAM files I then fed into QuEST using

/QuEST_2.4/generate_QuEST_parameters.pl -sam_align_ChIP $1  -sam_align_RX_noIP $2 -gt sk36_index.gt -ap  QuEST_analysis  -ChIP_name  $3

aligments: 0.02 M, matched : 0.00 M, no match: 0.00 M, not in gt: 0.009 M, offending: 0.000 M
aligments: 0.05 M, matched : 0.00 M, no match: 0.00 M, not in gt: 0.035 M, offending: 0.000 M
aligments: 0.07 M, matched : 0.00 M, no match: 0.01 M, not in gt: 0.062 M, offending: 0.000 M
aligments: 0.10 M, matched : 0.00 M, no match: 0.01 M, not in gt: 0.088 M, offending: 0.000 M
aligments: 0.13 M, matched : 0.00 M, no match: 0.02 M, not in gt: 0.106 M, offending: 0.000 M
aligments: 0.16 M, matched : 0.00 M, no match: 0.02 M, not in gt: 0.126 M, offending: 0.000 M
alig
aligments: 0.21 M, matched : 0.00 M, no match: 0.04 M, not in gt: 0.168 M, offending: 0.000 M
aligments: 0.24 M, matched : 0.00 M, no match: 0.04 M, not in gt: 0.186 M, offending: 0.000 M
aligments: 0.27 M, matched : 0.00 M, no match: 0.05 M, not in gt: 0.212 M, offending: 0.000 M

I'm using the bwa aligned sam files, and have created a simple -gt file which looks like this

chr 2388435

Thanks for any advisement!

Tara

[Anton]

Hi Tara, 

your genome table looks like a suspect. It doesn't have a chromosome number.

If your reads align to say, to chr3, which you can check with the following command:

cat your_alignments.sam | grep "chr3"

then your genome table should have the same chromosome name and include its name "chr3". For example, you should see a line in the genome table file that looks something like this:

chr3 9999999

Which organism is this? Perhaps you can post a few lines from your sam file and the entire genome table here so that I can point out the source of inconsistency.

Best, Anton

[Federico Gaiti]

Hi all,

I've been trying to run QuEST. I am working on a sponge species I have thousands of scaffolds which a good percentage are relatively short (<10Kb). 

The first trial with QuEST I did not remove the smaller scaffolds and I got peak shift estimate = NA. I then selected the recommended size of 50 and the script went on till the end without errors. 

Though, the QuEST analysis found 0 peaks in the output directories.

This is the command I used:

./QuEST_2.4/generate_QuEST_parameters.pl -sam_align_ChIP /opsin/u/federico/RIP/BAM/Ezh2/Ezh2_sorted_rmdup.sam -sam_align_RX_noIP/opsin/u/federico/RIP/BAM/Input/Input_sorted_rmdup.sam -gt chrom_correct.sizes -ap /opsin/u/federico/RIP/BAM/QuEST/ -ChIP_name Ezh2_RIP

The treatment SAM file looks like this: 

M01177:26:000000000-A8A2V:1:2101:23411:18636 147 Contig9999 2490 50 68M = 2490 -68 CAAACACCACAGTACTCCAATTGCTTGCCGTCCTATACAAGCAATTCAGAGGATAGCAGCTGAAGGAC ?GEFFEE9FGFFGGGGGGFGFF@F:EEGFFGFGGDGE@DADCCF,FGFFF8FE,E,GFEC9FF,CBA- AS:i:-3 XN:i:0 XM:i:1 XO:i:0XG:i:0 NM:i:1 MD:Z:55T12 YT:Z:UU NH:i:1 XS:A:+

The input SAM file looks like this:

M01177:26:000000000-A8A2V:1:1115:10786:9719 403 Contig9999 3905 0 38M = 3905 -38 CCAATAGTGATTGTACATGTATATCCTTTTGAGTGGGT GGGGGGGFGGGFFEFEGFGGGFGGGFGGGGGGGCCCCC AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:38 YT:Z:UU NH:i:29 XS:A:+ HI:i:28



The chromosome table file looks like this:

Contig13522 1888931
Contig13521 1425252
Contig13520 1092842
Contig13519 1050623
Contig13518 1034607
Contig13517 931383
Contig13516 899346
Contig13515 852899
Contig13514 817803
Contig13513 765180

I was wondering if you guys have any ideas about why I am getting 0 peaks accepted. Will removing the smaller scaffolds fix the issue? 

Thanks and let me know if I should provide additional information for debugging

Federico