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Technical and biological sources of noise confound multiplexed enhancer AAV screening.

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Paper

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The authors' code

Python · 106 lines · 2.7 KB · CC-BY-4.0

  1. #!/usr/bin/env python3
  3. import pysam
  4. import argparse
  5. from collections import defaultdict
  7. def parse_args():
  8. parser = argparse.ArgumentParser(
  9. description="Extract CB, UMI, and 8bp barcodes from reads in a BAM file"
  10. )
  11. parser.add_argument(
  12. "-b", "--bam",
  13. required=True,
  14. help="Input filtered BAM file"
  15. )
  16. parser.add_argument(
  17. "-o", "--out",
  18. required=True,
  19. help="Output TSV file"
  20. )
  21. parser.add_argument(
  22. "-k", "--barcode_length",
  23. type=int,
  24. default=8,
  25. help="Barcode length (default: 8)"
  26. )
  27. parser.add_argument(
  28. "--whitelist",
  29. default=None,
  30. help="Optional file with valid barcodes (one per line)"
  31. )
  32. parser.add_argument(
  33. "--debug",
  34. action="store_true",
  35. help="Print first 10 reads and kmers for debugging"
  36. )
  37. return parser.parse_args()
  40. def load_whitelist(path):
  41. with open(path) as f:
  42. return set(line.strip().upper() for line in f if line.strip())
  45. def extract_kmers(seq, k):
  46. """Return all k-length substrings (kmers) from a sequence"""
  47. return {seq[i:i+k] for i in range(len(seq) - k + 1)}
  50. def main():
  51. args = parse_args()
  53. whitelist = None
  54. if args.whitelist:
  55. whitelist = load_whitelist(args.whitelist)
  57. bam = pysam.AlignmentFile(args.bam, "rb")
  58. counts = defaultdict(int)
  60. debug_count = 0
  62. for read in bam.fetch(until_eof=True):
  64. # Skip unmapped / secondary / supplementary reads
  65. if read.is_unmapped or read.is_secondary or read.is_supplementary:
  66. continue
  68. # Get CB / UB tags
  69. try:
  70. cb = read.get_tag("CB").replace("-1", "")
  71. umi = read.get_tag("UB")
  72. except KeyError:
  73. continue
  75. seq = read.query_sequence
  76. if seq is None:
  77. continue
  79. seq = seq.upper()
  80. kmers = extract_kmers(seq, args.barcode_length)
  82. if whitelist is not None:
  83. kmers = kmers & whitelist
  85. for bc in kmers:
  86. counts[(cb, umi, bc)] += 1
  88. # Optional debugging: print first 10 reads
  89. if args.debug and debug_count < 10:
  90. print(f"Read: {seq}")
  91. print(f"CB: {cb}, UMI: {umi}, kmers: {kmers}")
  92. debug_count += 1
  94. bam.close()
  96. # Write output TSV
  97. with open(args.out, "w") as out:
  98. out.write("CB\tUMI\tbarcode8bp\tread_count\n")
  99. for (cb, umi, bc), n in counts.items():
  100. out.write(f"{cb}\t{umi}\t{bc}\t{n}\n")
  102. print(f"Done! Output written to {args.out}")
  105. if __name__ == "__main__":
  106. main()

extract_cb_umi_barcode_from_bam.py, under CC-BY-4.0 · at the source

Overview

Authors: Avery C Hunker1, John K Mich1, Naz Taskin1, Amy Torkelson1, Trangthanh Cardenas1, Jean-Benoît Lalanne2, Joseph T Mahoney1, Darren Bertagnolli1, Anish Bhaswanth Chakka1, Rushil Chakrabarty1, Nicholas P Donadio1, Rebecca Ferrer1, Molly Gasperini3, Jeff Goldy1, Junitta Guzman1, Kelly Jin1, Shannon Khem1, Rana Kutsal1, Refugio A Martinez1, Dakota Newman1
and 15 other authorsNick Pena1, Christine Rimorin1, Dana Rocha1, Nadiya V Shapovalova1, Michael Tieu1, Natalie Weed1, Thomas Zhou1, Rebecca Hodge1, Shenqin Yao1, Jay Shendure2,3,4,5,6, Kimberly A Smith1, Ed S Lein1,7, Bosiljka Tasic1, Boaz P Levi1, Jonathan T Ting1,8,9
ORCID iDs: Avery C Hunker, John K Mich, Amy Torkelson, Rushil Chakrabarty, Kelly Jin, Rana Kutsal, Natalie Weed, Rebecca Hodge, Shenqin Yao, Jay Shendure, Kimberly A Smith, Ed S Lein, Bosiljka Tasic, Boaz P Levi, Jonathan T Ting
  1. Allen Institute for Brain Science, Seattle, WA USA
  2. Department of Genome Sciences, University of Washington, Seattle, WA USA
  3. Seattle Hub for Synthetic Biology, Seattle, WA USA
  4. Brotman Baty Institute for Precision Medicine, Seattle, WA USA
  5. Howard Hughes Medical Institute, Seattle, WA USA
  6. Allen Discovery Center for Cell Lineage Tracing, Seattle, WA USA
  7. Department of Neurological Surgery, University of Washington, Seattle, WA USA
  8. Department of Neurobiology & Biophysics, University of Washington, Seattle, WA USA
  9. Washington National Biomedical Research Center, Seattle, WA USA
Institutions: Allen Institute for Brain Science (United States); University of Washington (United States); Howard Hughes Medical Institute (United States); Brotman Baty Institute (United States)
Journal: Nature communications, volume 17, issue 1, article 3738
Dates: received 29 May 2025; accepted 8 April 2026; published online 28 April 2026
Type: Research article · Language: English
License: CC BY-NC-ND
Identifiers: DOI 10.1038/s41467-026-72147-8 · PMID 42049725 · PMCID PMC13125632 · OpenAlex W4406457060
Open access: gold, a free copy (OpenAlex)
Preprint: doi:10.1101/2025.01.14.633018
Status: code verified
Categories: genetics / omics (modality), human (organism), mouse (organism), clinical / translational (subfield)
Methods: Evoked potentials
Keywords: Genetic vectors, Expression systems, Gene expression profiling, High-throughput screening
MeSH: Dependovirus*, Enhancer Elements, Genetic*, Genetic Vectors*, Animals, Brain, HEK293 Cells, Humans, Mice (* major topic)
Topic: Photoacoustic and Ultrasonic Imaging (Biomedical Engineering, Engineering), according to OpenAlex
Funding: National Institutes of Health (1uf1mh128339-01)
Citations: cited by 2 papers (Europe PMC); 60 references in the paper
Research resources: RRID:SCR_018099, RRID:SCR_024672, RRID:SCR_025643

Abstract

The abstract is not reproduced here: the paper's license (CC BY-NC-ND) does not allow it. Read it in the paper, at the publisher or on Europe PMC.

Repository

Its files are read in the Code ↔ Paper reader above.

Zenodo 18753067

License: CC-BY-4.0
State: the link answers, verified on 30 September 2026
Evidence: files inventoried
Size: 1 file
Software Heritage: not checked
Found in: “Code availability”
Not found: README, license file, CITATION.cff, environment file, tests, continuous integration, documentation
Tools: pysam (2 files)
Availability: 1 check, the latest on 30 September 2026: the link answers (HTTP 200)
  • 30 September 2026: the link answers (HTTP 200)
4 files
At the source:

Code availability statement

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  • it points to the authors' code: Zenodo 18753067

Read it in the paper: doi.org/10.1038/s41467-026-72147-8.

Tracing map

Proposed by the machine: these links were found in the paper and verified at the source, without human review. The map will receive a Zenodo DOI once one of the paper's authors has validated it with their ORCID.

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Its JSON (tracing-map.json) is deposited on Zenodo with its DOI once the map is validated.

Data

Datasets cited

Code and data availability statement

The paper has a code and data availability statement. Its license (CC BY-NC-ND) does not allow reproducing it here; in short, from what the harvester recognized in it:

Read it in the paper: doi.org/10.1038/s41467-026-72147-8.

Versions

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Version 1, 30 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 35 authors, 4 keywords, 8 MeSH terms, 1 funder, 57 references, 3 RRIDs.

Cite

This paper

Hunker, A. C., Mich, J. K., Taskin, N., Torkelson, A., Cardenas, T., Lalanne, J.-B., Mahoney, J. T., Bertagnolli, D., Chakka, A. B., Chakrabarty, R., Donadio, N. P., Ferrer, R., Gasperini, M., Goldy, J., Guzman, J., Jin, K., Khem, S., Kutsal, R., Martinez, R. A., . . . Ting, J. T. (2026). Technical and biological sources of noise confound multiplexed enhancer AAV screening. Nature communications, 17(1), 3738. https://doi.org/10.1038/s41467-026-72147-8

BibTeX

@article{hunker2026technical,
author = {Hunker, Avery C and Mich, John K and Taskin, Naz and Torkelson, Amy and Cardenas, Trangthanh and Lalanne, Jean-Benoît and Mahoney, Joseph T and Bertagnolli, Darren and Chakka, Anish Bhaswanth and Chakrabarty, Rushil and Donadio, Nicholas P and Ferrer, Rebecca and Gasperini, Molly and Goldy, Jeff and Guzman, Junitta and Jin, Kelly and Khem, Shannon and Kutsal, Rana and Martinez, Refugio A and Newman, Dakota and Pena, Nick and Rimorin, Christine and Rocha, Dana and Shapovalova, Nadiya V and Tieu, Michael and Weed, Natalie and Zhou, Thomas and Hodge, Rebecca and Yao, Shenqin and Shendure, Jay and Smith, Kimberly A and Lein, Ed S and Tasic, Bosiljka and Levi, Boaz P and Ting, Jonathan T},
title = {{Technical and biological sources of noise confound multiplexed enhancer AAV screening}},
journal = {Nature communications},
year = {2026},
month = apr,
volume = {17},
number = {1},
pages = {3738},
publisher = {Nature Publishing Group},
issn = {2041-1723},
doi = {10.1038/s41467-026-72147-8},
url = {https://doi.org/10.1038/s41467-026-72147-8},
pmid = {42049725},
pmcid = {PMC13125632}
}

RIS

TY - JOUR
AU - Hunker, Avery C
AU - Mich, John K
AU - Taskin, Naz
AU - Torkelson, Amy
AU - Cardenas, Trangthanh
AU - Lalanne, Jean-Benoît
AU - Mahoney, Joseph T
AU - Bertagnolli, Darren
AU - Chakka, Anish Bhaswanth
AU - Chakrabarty, Rushil
AU - Donadio, Nicholas P
AU - Ferrer, Rebecca
AU - Gasperini, Molly
AU - Goldy, Jeff
AU - Guzman, Junitta
AU - Jin, Kelly
AU - Khem, Shannon
AU - Kutsal, Rana
AU - Martinez, Refugio A
AU - Newman, Dakota
AU - Pena, Nick
AU - Rimorin, Christine
AU - Rocha, Dana
AU - Shapovalova, Nadiya V
AU - Tieu, Michael
AU - Weed, Natalie
AU - Zhou, Thomas
AU - Hodge, Rebecca
AU - Yao, Shenqin
AU - Shendure, Jay
AU - Smith, Kimberly A
AU - Lein, Ed S
AU - Tasic, Bosiljka
AU - Levi, Boaz P
AU - Ting, Jonathan T
TI - Technical and biological sources of noise confound multiplexed enhancer AAV screening
T2 - Nature communications
J2 - Nat Commun
PY - 2026
DA - 2026/04/28
VL - 17
IS - 1
SP - 3738
SN - 2041-1723
PB - Nature Publishing Group
DO - 10.1038/s41467-026-72147-8
UR - https://doi.org/10.1038/s41467-026-72147-8
LA - en
ER -

CSL-JSON

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