original ST barcode

[mandexue]

Dear SpatialTE developers,

I hope you are doing well.

I am currently trying to perform the TE analysis from this paper:

SpatialTE paper in Cell

Specifically, I am working with dataset:

SRR11832808
GSE144239

I believe this dataset is an original Spatial Transcriptomics (ST) dataset. The GEO submission provides a barcode file named:

GSE144239_ST_barcode_id.txt

The barcode file looks like this:

GTCCGATATGATTGCCGC 2 6
GCGACGCGGCATAGTTTA 2 8
ACGTGCGCAAATATGACG 2 10
...

However, when I run SpatialTE, I encounter the following errors:

Error running the pipeline
object of type 'NoneType' has no len()

Then downstream steps fail because several BAM files are not generated:

[E::hts_open_format] Failed to open file "annotated.bam" : No such file or directory
samtools view: failed to open "annotated.bam" for reading: No such file or directory

I also see:

samtools view: Could not read file "mm10_all_from_ucsc.bed": No such file or directory
Error: Unable to open file mm10_all_from_ucsc.bed. Exiting.

and later:

RuntimeError: Error during annotation, input file not present annotated_overlappingTE.bam

followed by additional missing intermediate BAM errors.

My questions are:

Does SpatialTE fully support the original ST barcode format used in GSE144239?
Is the GSE144239_ST_barcode_id.txt format correct for SpatialTE input?

Could the initial:

object of type 'NoneType' has no len()

error indicate a barcode parsing issue?

Should I convert the barcode file into another format before running the pipeline?
Is the missing mm10_all_from_ucsc.bed file related to the primary error, or is it a separate configuration issue?

Any guidance would be greatly appreciated.

Thank you very much for your help and for developing SpatialTE.

Best regards,
Mande

[bvaldebenitom]

Hi! Thanks for reaching out. I checked the paper, and I am not aware of it using SpatialTE. You are right however that it is from the original ST technology. To run SpatialTE, the barcode files must be tab-delimited, and yours does not look like that. Also, I can see that the data you mention is from human, and you are referencing the mouse TE BED file. Can you share the configuration file you are using? Also, can you confirm that you have STPipeline working and running? Best, Braulio. El lun, 25 may 2026 a las 19:01, Mande Xue ***@***.***>) escribió:

…

*mandexue* created an issue (bvaldebenitom/SpatialTE#4) <#4> Dear SpatialTE developers, I hope you are doing well. I am currently trying to reproduce the analysis from your paper: SpatialTE paper in Cell <https://www.sciencedirect.com/science/article/pii/S0092867420306723?utm_source=chatgpt.com> Specifically, I am working with dataset: SRR11832808 GSE144239 I believe this dataset is an original Spatial Transcriptomics (ST) dataset. The GEO submission provides a barcode file named: GSE144239_ST_barcode_id.txt The barcode file looks like this: GTCCGATATGATTGCCGC 2 6 GCGACGCGGCATAGTTTA 2 8 ACGTGCGCAAATATGACG 2 10 ... However, when I run SpatialTE, I encounter the following errors: Error running the pipeline object of type 'NoneType' has no len() Then downstream steps fail because several BAM files are not generated: [E::hts_open_format] Failed to open file "annotated.bam" : No such file or directory samtools view: failed to open "annotated.bam" for reading: No such file or directory I also see: samtools view: Could not read file "mm10_all_from_ucsc.bed": No such file or directory Error: Unable to open file mm10_all_from_ucsc.bed. Exiting. and later: RuntimeError: Error during annotation, input file not present annotated_overlappingTE.bam followed by additional missing intermediate BAM errors. My questions are: Does SpatialTE fully support the original ST barcode format used in GSE144239? Is the GSE144239_ST_barcode_id.txt format correct for SpatialTE input? Could the initial: object of type 'NoneType' has no len() error indicate a barcode parsing issue? Should I convert the barcode file into another format before running the pipeline? Is the missing mm10_all_from_ucsc.bed file related to the primary error, or is it a separate configuration issue? Any guidance would be greatly appreciated. Thank you very much for your help and for developing SpatialTE. Best regards, Mande — Reply to this email directly, view it on GitHub <#4?email_source=notifications&email_token=AHOPFKNFKSVXBEJDO6RDTAD44TGGLA5CNFSL4Z3JMQ5C6L3HNF2C22DVMIXUS43TOVSS6NBVGE4TSOBVGMZDDJTSMVQXG33OVJZXKYTTMNZGSYTFMSSWK5TFNZ2KYZTPN52GK4S7MNWGSY3L>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AHOPFKND7NYKWGGL47ZFTTD44TGGLAVCNFSM6AAAAACZMZVR5KVHI2DSMVQWIX3LMV43ASLTON2WKOZUGUYTSOJYGUZTEMI> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[mandexue]

Hi Braulio,

Thanks for your reply and for pointing that out. I just want to use this dataset for some TE analysis using SpatialTE, since it appears to come from the original ST technology.

I checked my barcode file again, and it is tab-delimited. The ^I symbols shown by cat -T correspond to tab characters. An example is shown below:

You are also correct that I mistakenly referenced the mouse TE BED file while working with human data. I have switched to the correct human TE annotation.

I do have STPipeline running successfully. I am attaching/sharing the configuration file I am currently using below for reference.

Unfortunately, I am still getting an error when running the pipeline.
enter the following:
eval "$(/risapps/rhel8/miniforge3/24.5.0-0/bin/conda shell.bash hook)"
Error running the pipeline
object of type 'NoneType' has no len()
[E::hts_open_format] Failed to open file "annotated.bam" : No such file or directory
samtools view: failed to open "annotated.bam" for reading: No such file or directory
[E::hts_open_format] Failed to open file "annotated_nofeature.bam" : No such file or directory
samtools view: failed to open "annotated_nofeature.bam" for reading: No such file or directory
Error: Unable to open file annotated_overlappingTE.bam. Exiting.
Error during annotation, input file not present annotated_overlappingTE.bam

Traceback (most recent call last):
File "//rsrch8/home/canbio/mxue/release_1.0/annotate_reads_with_TEs.py", line 32, in
annotateReads(input_file,
File "/rsrch8/home/canbio/mxue/.conda/envs/SpatialTE/lib/python3.8/site-packages/stpipeline/core/annotation.py", line 243, in annotateReads
raise RuntimeError(error)
RuntimeError: Error during annotation, input file not present annotated_overlappingTE.bam

[E::hts_open_format] Failed to open file "annotated_overlappingTE_withXF.bam" : No such file or directory
samtools view: failed to open "annotated_overlappingTE_withXF.bam" for reading: No such file or directory

---

***** ERROR: Requested column 2, but database file - only has fields 1 - 0.
Error: Unable to open file annotated_overlappingTE_withXF.bam. Exiting.
[E::hts_open_format] Failed to open file "annotated_overlappingTE_withXF.bam" : No such file or directory
samtools view: failed to open "annotated_overlappingTE_withXF.bam" for reading: No such file or directory
Error creating dataset, input file not present selected_TE_ms100.bam

Traceback (most recent call last):
File "/rsrch8/home/canbio/mxue/release_1.0/create_final_output.py", line 31, in
createDataset(input_bam,
File "/rsrch8/home/canbio/mxue/.conda/envs/SpatialTE/lib/python3.8/site-packages/stpipeline/common/dataset.py", line 96, in createDataset
raise RuntimeError(error)
RuntimeError: Error creating dataset, input file not present selected_TE_ms100.bam

Error during annotation, input file not present annotated_overlappingTE_withXF.bam

Traceback (most recent call last):
File "/rsrch8/home/canbio/mxue/release_1.0/annotate_reads_with_TEs.py", line 32, in
annotateReads(input_file,
File "/rsrch8/home/canbio/mxue/.conda/envs/SpatialTE/lib/python3.8/site-packages/stpipeline/core/annotation.py", line 243, in annotateReads
raise RuntimeError(error)
RuntimeError: Error during annotation, input file not present annotated_overlappingTE_withXF.bam

Error creating dataset, input file not present annotated_overlappingTE_withXF_MS0.bam

Traceback (most recent call last):
File "/rsrch8/home/canbio/mxue/release_1.0/create_final_output.py", line 31, in
createDataset(input_bam,
File "/rsrch8/home/canbio/mxue/.conda/envs/SpatialTE/lib/python3.8/site-packages/stpipeline/common/dataset.py", line 96, in createDataset
raise RuntimeError(error)
RuntimeError: Error creating dataset, input file not present annotated_overlappingTE_withXF_MS0.bam
Best,
Mande

[bvaldebenitom]

Hi @mandexue ,

apologies for the delay. Can you share the first few lines of your TE BED file?

Since the data is public, I will test it soon to see if I am able to replicate the issues.

[mandexue]

Hi Braulio,

Thanks a lot! Fortunately, I was able to get the analysis working using SoloTE, which I understand is another tool you developed. I really appreciate your help, and I want to thank you for creating such a great tool.

Best,
Mande

[bvaldebenitom]

That is very great to hear, and you are right!! Me and several others have successfully used SoloTE for Spatial data indeed!

All the best,
Braulio.