AssertionError: slocal can't be smaller than d! #171
Comments
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You used the default slocal value 1000bp. Your mean fragment length (d)
is 1109bp. That's why it complains "slocal can't be smaller than d!". I
think you can get through by setting --slocal to a value larger than
1109, eg. 2000.
In addition, it seems to me that the fragment length is very long (for a
ChIP-seq experiment). Just curious about (1) Is it really ChIP-seq
data? (2) How was the library sequenced? on what platform? (The fragment
length seems too long for PE sequencing on Hiseq. Or, I am behind the
times?)
Best,
Dejian
…On 1/17/17 5:10 PM, JP Cartailler wrote:
Greetings,
(Googling around wasn't too helpful, so I hope posting here is
appropriate).
I'm trying to run a "simple" MACS2 job (MACS2 2.1.0 20150420 rc):
|macs2 callpeak \ --name 'HA vs. WT' \ --treatment $CHIPDATA1 \
--control $CHIPCTRL1 \ --outdir $DIRECTORY \ --qvalue .01 \ --format
BAMPE \ --gsize mm \ --tempdir $TMP_DIRECTORY |
The output of the job is as follows:
|Log -- Running PEAKCALL_MACS2 Job Log -- ================= Log --
Created tmp directory INFO @ Tue, 17 Jan 2017 15:11:11: # Command
line: callpeak --name HA vs. WT --treatment
/home/user/data/Processed-Data/3259-MAM/05-samtools/Ali
gned_sample_3259-MAM-7.out.sorted.bam --control
/home/user/data/Processed-Data/3259-MAM/05-samtools/Aligned_sample_32
59-MAM-8.out.sorted.bam --outdir 07-peakcall_macs2 --qvalue .01
--format BAMPE --gsize mm --tempdir 07-peakcall_macs2/te mp #
ARGUMENTS LIST: # name = Neurog3-HA vs. WT # format = BAMPE # ChIP-seq
file =
['/home/user/data/Processed-Data/3259-MAM/05-samtools/Aligned_sample_3259-MAM-7.out.sorted.bam']
# control file =
['/home/user/data/Processed-Data/3259-MAM/05-samtools/Aligned_sample_3259-MAM-8.out.sorted.bam']
# effective genome size = 1.87e+09 # band width = 300 # model fold =
[5, 50] # qvalue cutoff = 1.00e-02 # Larger dataset will be scaled
towards smaller dataset. # Range for calculating regional lambda is:
1000 bps and 10000 bps # Broad region calling is off # Paired-End mode
is on INFO @ Tue, 17 Jan 2017 15:11:11: #1 read fragment files... INFO
@ Tue, 17 Jan 2017 15:11:11: #1 read treatment fragments... INFO @
Tue, 17 Jan 2017 15:11:30: 1000000 INFO @ Tue, 17 Jan 2017 15:11:48:
2000000 INFO @ Tue, 17 Jan 2017 15:12:06: 3000000 ... ... ... INFO @
Tue, 17 Jan 2017 15:44:11: 42000000 INFO @ Tue, 17 Jan 2017 15:44:37:
43000000 INFO @ Tue, 17 Jan 2017 15:45:23: #1 mean fragment size is
determined as 1109 bp from treatment INFO @ Tue, 17 Jan 2017 15:45:24:
#1 note: mean fragment size in control is 1853 bp -- value ignored
INFO @ Tue, 17 Jan 2017 15:45:24: #1 fragment size = 1109 INFO @ Tue,
17 Jan 2017 15:45:24: #1 total fragments in treatment: 42552760 INFO @
Tue, 17 Jan 2017 15:45:24: #1 user defined the maximum fragments...
INFO @ Tue, 17 Jan 2017 15:45:24: #1 filter out redundant fragments by
allowing at most 1 identical fragment(s) INFO @ Tue, 17 Jan 2017
15:48:03: #1 fragments after filtering in treatment: 31036085 INFO @
Tue, 17 Jan 2017 15:48:03: #1 Redundant rate of treatment: 0.27 INFO @
Tue, 17 Jan 2017 15:48:03: #1 total fragments in control: 43232586
INFO @ Tue, 17 Jan 2017 15:48:03: #1 user defined the maximum
fragments... INFO @ Tue, 17 Jan 2017 15:48:03: #1 filter out redundant
fragments by allowing at most 1 identical fragment(s) INFO @ Tue, 17
Jan 2017 15:50:45: #1 fragments after filtering in control: 29065439
INFO @ Tue, 17 Jan 2017 15:50:45: #1 Redundant rate of control: 0.33
INFO @ Tue, 17 Jan 2017 15:50:45: #1 finished! INFO @ Tue, 17 Jan 2017
15:50:45: #2 Build Peak Model... INFO @ Tue, 17 Jan 2017 15:50:45: #2
Skipped... INFO @ Tue, 17 Jan 2017 15:50:45: #2 Use 1109 as fragment
length INFO @ Tue, 17 Jan 2017 15:50:45: #3 Call peaks... Traceback
(most recent call last): File "/home/user/bin/anaconda2/bin/macs2",
line 617, in <module> main() File
"/home/user/bin/anaconda2/bin/macs2", line 57, in main run( args )
File
"/home/user/bin/anaconda2/lib/python2.7/site-packages/MACS2/callpeak_cmd.py",
line 264, in run peakdetect.call_peaks() File "MACS2/PeakDetect.pyx",
line 105, in MACS2.PeakDetect.PeakDetect.call_peaks
(MACS2/PeakDetect.c:1632) File "MACS2/PeakDetect.pyx", line 177, in
MACS2.PeakDetect.PeakDetect.__call_peaks_w_control
(MACS2/PeakDetect.c:2163) AssertionError: slocal can't be smaller than
d! |
Any ideas on what I might be doing wrong? Running out of ideas...
Thanks!!!
JP
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Dejian - thanks very much for helping me understand the error/log combination. Indeed, the fragment length seems very long. It is a ChIP-Seq experiment and after checking the library QC gel, the fragments are ~300bp. Precipitated chromatin was used to prepare sequencing libraries using NEB DNA Ultra II reagents. Samples were sequenced at PE-75 using HiSeq 3000, targeting 30M reads per sample. So something must be wrong upstream IMO. After the usual quality control/trimming, I aligned our reads via STAR. I did find a reference that indicates that I need to look at my alignment parameters more closely for ChIP-Seq data/STAR. I'll report back for others, in case I'm not the only to run into this issue. Again, many thanks! Very helpful! |
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I read the reference you provided. Very interesting discussion. But I
prefer bowtie2 rather than STAR for aligning ChIP-seq reads. Good luck!
-Dejian
…On 1/18/17 1:59 PM, JP Cartailler wrote:
Dejian - thanks very much for helping me understand the error/log
combination.
Indeed, the fragment length seems very long. It /is/ a ChIP-Seq
experiment and after checking the library QC gel, the fragments are
~300bp. Precipitated chromatin was used to prepare sequencing
libraries using NEB DNA Ultra II reagents. Samples were sequenced at
PE-75 using HiSeq 3000, targeting 30M reads per sample.
So something must be wrong upstream IMO. After the usual quality
control/trimming, I aligned our reads via STAR. I did find a reference
<https://groups.google.com/d/msg/rna-star/E_mKqm9jDm0/ZINystrdWRQJ>
that indicates that I need to look at my alignment parameters more
closely for ChIP-Seq data/STAR. I'll report back for others, in case
I'm not the only to run into this issue.
Again, many thanks! Very helpful!
JP
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I read the reference you provided. Very interesting discussion. But I prefer bowtie2 rather than STAR for aligning ChIP-seq reads. Good luck! -Dejian
On 1/18/17 1:59 PM, JP Cartailler wrote:
Dejian - thanks very much for helping me understand the error/log combination.
Indeed, the fragment length seems very long. It is a ChIP-Seq experiment and after checking the library QC gel, the fragments are ~300bp. Precipitated chromatin was used to prepare sequencing libraries using NEB DNA Ultra II reagents. Samples were sequenced at PE-75 using HiSeq 3000, targeting 30M reads per sample.
So something must be wrong upstream IMO. After the usual quality control/trimming, I aligned our reads via STAR. I did find a reference<https://groups.google.com/d/msg/rna-star/E_mKqm9jDm0/ZINystrdWRQJ> that indicates that I need to look at my alignment parameters more closely for ChIP-Seq data/STAR. I'll report back for others, in case I'm not the only to run into this issue.
Again, many thanks! Very helpful!
JP
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Greetings,
(Googling around wasn't too helpful, so I hope posting here is appropriate).
I'm trying to run a "simple" MACS2 job (MACS2 2.1.0 20150420 rc):
The output of the job is as follows:
Any ideas on what I might be doing wrong? Running out of ideas...
Thanks!!!
JP
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