Begin v3 with IGVF uniform pipeline processed data

[adamklie]

Context

For igvf-data/igvf_sc-islet_10X-Multiome, start v3 using IGVF uniform pipeline.

Tasks

• Sort out the R2 barcode issue for element samples
• Download new data and run sample QC
• Run integration and peak calling
• Run differential analysis (once methodology is finalized)

[adamklie]

Dependencies

• Blocked by Finalize DESeq2 pseudobulk methodology for RNA and ATAC #4 (needs finalized DESeq2 methodology to apply to v3)

[adamklie]

Started to handle the barcode issue here igvf-data/igvf_sc-islet_10X-Multiome/scratch/2026_03_18

The 20 element samples R2 files have been reset. We now need to reupload the rev comped barcodes using igvf_utils

We also need to create some reproducible code that does the whole fix here: igvf-data/igvf_sc-islet_10X-Multiome/bin/10_submission. It's a similar situation to the padded Ns thing (more info here: igvf-data/igvf_sc-islet_10X-Multiome/scratch/2026_02_23)

[adamklie]

Implementation: R2 Barcode Fix + Submission Documentation

Problem

10 samples sequenced on Element AVITI have ATAC R2 barcodes in forward orientation. The IGVF uniform pipeline expects reverse complement (Illumina default). These 10 samples fail during barcode matching.

What was done

1. Submission documentation (igvf-data/SUBMISSION.md)

• Full guide to the 10X Multiome IGVF portal submission pipeline
• Covers all 5 steps in bin/10_submission/, plus barcode fixes
• Documents common issues (N-padding, R2 orientation) and how to diagnose/fix
• Portal API reference and metadata hierarchy

2. Revcomp fix added to 3_portal_submission_tables.ipynb

• Added a new section "Handling R2 barcode orientation for AVITI-sequenced samples" following the same pattern as the existing N-padding fix
• Creates sequence_files_revcomp.txt with both N-padding AND revcomp fixes applied
• Uses the already-generated revcomped FASTQs at fastq/patched_revcomp/ (20 files)
• Calculates new MD5 checksums and updates aliases with _revcomp suffix

Files modified/created

• igvf-data/SUBMISSION.md — new submission documentation
• bin/10_submission/3_portal_submission_tables.ipynb — added 6 cells for revcomp fix (cells 69-74)

Next steps

• Run the updated notebook to generate sequence_files_revcomp.txt
• Upload patched files to IGVF portal (coordinate with DACC if needed)
• Request pipeline re-run on the 10 affected analysis sets

Reference

• Exploratory work: scratch/2026_03_18/confirm_and_fix_barcodes.ipynb
• N-padding precedent: scratch/2026_02_23/
• Patched files: fastq/patched_revcomp/ (20 files, 10 samples × 2 lanes)

[adamklie]

Status Update (2026-03-23)

R2 Barcode Fix — COMPLETED

• Confirmed barcode orientation issue across all 54 samples (10 AVITI = Forward, 44 Illumina = RevComp Last 16)
• Reverse-complemented FASTQs generated (20 files at fastq/patched_revcomp/)
• Barcode verification: 20/20 PASS against seqspec onlist (IGVFFI7587TJLC, 736k barcodes)
• Sample swap check: every revcomp file's first read = RC(original first read)
• Portal upload: 20/20 files re-uploaded to existing accessions via conn.upload_file()
• All files were upload_status: invalidated, status: in progress — eligible for re-upload

Submission Documentation

• Created igvf-data/SUBMISSION.md — full 10X Multiome submission pipeline guide
• Added revcomp section to bin/10_submission/3_portal_submission_tables.ipynb

Files created

• scratch/2026_03_23/barcode_verification/verify_revcomp_barcodes.py — verification script
• scratch/2026_03_23/barcode_verification/barcode_verification_results.csv — results
• scratch/2026_03_23/barcode_verification/reupload_revcomp_files.py — upload script
• scratch/2026_03_23/barcode_verification/2026_03_23_portal_submission.sh — submission wrapper
• igvf-data/SUBMISSION.md — documentation

Remaining for v3

• Wait for portal validation of re-uploaded files
• Request uniform pipeline re-run on 10 affected analysis sets
• Download processed data and run sample QC
• Integration, peak calling, differential analysis on v3 data