# ATAC-seq Analysis Using MACS2 Callpeak **URL:** \ **Category:** usegalaxy.org support\ **Tags:** troubleshooting, picard\_markduplicates\ **Created:** [November 12, 2024, 4:08pm UTC](https://help.galaxyproject.org/t/atac-seq-analysis-using-macs2-callpeak/13886 "2024-11-12T16:08:52Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![shgalaxyuser](https://avatars.discourse-cdn.com/v4/letter/s/3bc359/32.png) [@shgalaxyuser](https://help.galaxyproject.org/u/shgalaxyuser)\ **Post date:** [November 12, 2024, 4:08pm UTC](https://help.galaxyproject.org/t/atac-seq-analysis-using-macs2-callpeak/13886/1 "2024-11-12T16:08:52Z")
Hey! I am currently trying to go through the ATAC-Seq Galaxy tutorial ([Hands-on: ATAC-Seq data analysis / ATAC-Seq data analysis / Epigenetics](https://training.galaxyproject.org/training-material/topics/epigenetics/tutorials/atac-seq/tutorial.html#trimming-reads)) and I am on the MACS2 callpeak step. I received an error and I am unsure of how to fix it. Any insight would be greatly appreciated! Here is what I input: | Number | 73 | | --- | --- | | Name | MACS2 callpeak on data 62 (Peaks in tabular format) | | Created | Tuesday Nov 12th 9:19:05 2024 GMT-5 | | Filesize | **-** | | Dbkey | mm10 | | Format | tabular | | File contents | [contents](https://usegalaxy.org/api/histories/bbd44e69cb8906b5392a7444d65eb960/contents/f9cad7b01a4721354ef60effa34ef599/display) | | History Content API ID | f9cad7b01a4721354ef60effa34ef599 | | History API ID | bbd44e69cb8906b5392a7444d65eb960 | | UUID | 858aa35b-c60e-49d3-bcf7-54024dba6a08 | | Full Path | /corral4/main/objects/8/5/8/dataset\_858aa35b-c60e-49d3-bcf7-54024dba6a08.dat | ## Tool Parameters | Input Parameter | Value | | --- | --- | | Are you pooling Treatment Files? | 0 | | ChIP-Seq Treatment File | 62: MarkDuplicates on data 57: BAM (as BED) | #SRR891268\_R1 #SRR891268\_R2| |Do you have a Control File?|0| |Format of Input Files|Single-end BAM| |Effective genome size|1870000000| |Build Model|nomodel| |Set extension size|200| |Set shift size|-100| |Peak detection based on|qvalue| |Minimum FDR (q-value) cutoff for peak detection|0.05| |Additional Outputs|Peaks as tabular file (compatible wih MultiQC) Peak summits Scores in bedGraph files (–bdg)| |advanced\_options|| |When set, scale the small sample up to the bigger sample|0| |Use fixed background lambda as local lambda for every peak region|0| |Save signal per million reads for fragment pileup profiles|0| |When set, use a custom scaling ratio of ChIP/control (e.g. calculated using NCIS) for linear scaling|Not available.| |The small nearby region in basepairs to calculate dynamic lambda|Not available.| |The large nearby region in basepairs to calculate dynamic lambda|Not available.| |Composite broad regions|nobroad| |Use a more sophisticated signal processing approach to find subpeak summits in each enriched peak region|1| |How many duplicate tags at the exact same location are allowed?|all| |Minimum fragment size in basepair|20| |Buffer size|100000| Here is the error I received: | Job State | error | | --- | --- | | Command Line | export PYTHON\_EGG\_CACHE=`pwd` && (macs2 callpeak -t ‘/corral4/main/objects/e/8/c/dataset\_e8caa316-80eb-411d-8233-08819f152174.dat’ --name MarkDuplicates\_on\_data\_57\_\_BAM\_\_as\_BED\_ --format BAM --gsize ‘1870000000’ --call-summits --keep-dup ‘all’ --d-min 20 --buffer-size 100000 --bdg --qvalue ‘0.05’ --nomodel --extsize ‘200’ --shift ‘-100’ 2\>&1 \> macs2\_stderr) && cp MarkDuplicates\_on\_data\_57\_\_BAM\_\_as\_BED\_\_peaks.xls ‘/corral4/main/jobs/062/436/62436865/outputs/dataset\_858aa35b-c60e-49d3-bcf7-54024dba6a08.dat’ && exit\_code\_for\_galaxy=$? && cat macs2\_stderr 2\>&1 && (exit $exit\_code\_for\_galaxy) | | Tool Standard Output | INFO @ Tue, 12 Nov 2024 14:19:09: # Command line: callpeak -t /corral4/main/objects/e/8/c/dataset\_e8caa316-80eb-411d-8233-08819f152174.dat --name MarkDuplicates\_on\_data\_57\_\_BAM\_\_as\_BED\_ --format BAM --gsize 1870000000 --call-summits --keep-dup all --d-min 20 --buffer-size 100000 --bdg --qvalue 0.05 --nomodel --extsize 200 --shift -100 # ARGUMENTS LIST: # name = MarkDuplicates\_on\_data\_57\_\_BAM\_\_as\_BED\_ # format = BAM # ChIP-seq file = [‘/corral4/main/objects/e/8/c/dataset\_e8caa316-80eb-411d-8233-08819f152174.dat’] # control file = None # effective genome size = 1.87e+09 # band width = 300 # model fold = [5, 50] # qvalue cutoff = 5.00e-02 # The maximum gap between significant sites is assigned as the read length/tag size. # The minimum length of peaks is assigned as the predicted fragment length “d”. # Larger dataset will be scaled towards smaller dataset. # Range for calculating regional lambda is: 10000 bps # Broad region calling is off # Paired-End mode is off # Searching for subpeak summits is on INFO @ Tue, 12 Nov 2024 14:19:09: #1 read tag files… INFO @ Tue, 12 Nov 2024 14:19:09: #1 read treatment tags… struct.error: unpack requires a buffer of 4 bytes Exception ignored in: ‘MACS2.IO.Parser.BAMParser.tsize’ Traceback (most recent call last): File “/usr/local/lib/python3.10/site-packages/MACS2/callpeak\_cmd.py”, line 389, in load\_tag\_files\_options ttsize = tp.tsize() struct.error: unpack requires a buffer of 4 bytes Traceback (most recent call last): File “/usr/local/bin/macs2”, line 653, in main() File “/usr/local/bin/macs2”, line 51, in main run( args ) File “/usr/local/lib/python3.10/site-packages/MACS2/callpeak\_cmd.py”, line 65, in run else: (treat, control) = load\_tag\_files\_options (options) File “/usr/local/lib/python3.10/site-packages/MACS2/callpeak\_cmd.py”, line 391, in load\_tag\_files\_options treat = tp.build\_fwtrack() File “MACS2/IO/Parser.pyx”, line 1169, in MACS2.IO.Parser.BAMParser.build\_fwtrack File “MACS2/IO/Parser.pyx”, line 1181, in MACS2.IO.Parser.BAMParser.build\_fwtrack File “MACS2/IO/Parser.pyx”, line 1166, in MACS2.IO.Parser.BAMParser.get\_references struct.error: unpack requires a buffer of 4 bytes | | Tool Standard Error | _empty_ | | Tool Exit Code | 1 | | Job Messages | Job Message 1: | - **desc:** Fatal error: Exit code 1 () - **error\_level:** 3 - **exit\_code:** 1 - **type:** exit\_code * * * Job Message 2: - **desc:** Fatal error: Matched on error: - **error\_level:** 3 - **match:** error: - **stream:** stdout - **type:** regex| Thank you so much! ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [November 13, 2024, 5:35pm UTC](https://help.galaxyproject.org/t/atac-seq-analysis-using-macs2-callpeak/13886/2 "2024-11-13T17:35:26Z")
Consolidated into here: [ATAC-Seq Analysis Using MACS2 Callpeak - #2 by jennaj](https://help.galaxyproject.org/t/atac-seq-analysis-using-macs2-callpeak/13888/2) For any one else sharing data, a history share link is best! [How to get faster help with your question](https://help.galaxyproject.org/docs?topic=11469) ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [November 13, 2024, 5:35pm UTC](https://help.galaxyproject.org/t/atac-seq-analysis-using-macs2-callpeak/13886/3 "2024-11-13T17:35:29Z")