# Updated RNAseq pipeline - AWG review needed

**URL:** <https://awg.osdr.space/t/updated-rnaseq-pipeline-awg-review-needed/1780>\
**Category:** OSDR Feedback\
**Tags:** omics, rna-seq, pipeline-update\
**Created:** [February 4, 2025, 5:40am UTC](https://awg.osdr.space/t/updated-rnaseq-pipeline-awg-review-needed/1780 "2025-02-04T05:40:29Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![asaravia](https://sea1.discourse-cdn.com/flex001/user_avatar/awg.osdr.space/asaravia/32/290_2.png) [@asaravia](https://awg.osdr.space/u/asaravia)\
**Post date:** [February 4, 2025, 5:40am UTC](https://awg.osdr.space/t/updated-rnaseq-pipeline-awg-review-needed/1780/1 "2025-02-04T05:40:29Z")

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Hi AWG members,  
I’m excited to announce that the [updated (bulk) RNAseq pipeline for eukaryotic organisms](https://github.com/nasa/GeneLab_Data_Processing/blob/DEV_RNAseq_vG/RNAseq/Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md) is ready for your review. Please click on the link to review the pipeline and **provide your feedback ASAP and no later than Monday, 2/17/2025**.

Updates include the following:

Updated [Ensembl Reference Files](https://github.com/nasa/GeneLab_Data_Processing/blob/DEV_RNAseq_vG/GeneLab_Reference_Annotations/Pipeline_GL-DPPD-7110_Versions/GL-DPPD-7110-A/GL-DPPD-7110-A_annotations.csv) to the following releases:

- Animals: Ensembl release 112
- Plants: Ensembl plants release 59
- Bacteria: Ensembl bacteria release 59

Software version updates.

STAR Alignment:

- Added unaligned reads FASTQ output file(s) via STAR `-outReadsUnmapped Fastq`:
  - {sample}\_Unmapped.out.mate1
  - {sample}\_Unmapped.out.mate2

RSeQC Analysis:

- Updated inner\_distance.py invocation to use a lower minimum value to account for longer read lengths
  - Previously used fixed -150 minimum value
  - Now uses -(max read length)

DESeq2 Analysis Workflow:

- Added variance-stabilizing transformation (VST) transformed counts output file, VST\_Counts\_GLbulkRNAseq.csv.
- Account for technical replicates
  - For datasets with uniform technical replicates (all samples have the same number of technical replicates):
    - Sum counts across technical replicates using DESeq2’s collapseReplicates

  - For datasets with non-uniform technical replicates:
    - Keep only the first N technical replicates for each sample, where N is the smallest number of technical replicates among all samples
    - Sum counts across the kept technical replicates

- Removed DGE and PCA output tables previously used for GeneLab visualization (visualization\_output\_table\_GLbulkRNAseq.csv and visualization\_PCA\_table\_GLbulkRNAseq.csv)
- ERCC-normalized DGE analysis was removed. The following output files were removed:
  - ERCC\_Normalized\_Counts\_GLbulkRNAseq.csv
  - ERCCnorm\_differential\_expression\_GLbulkRNAseq.csv
  - ERCCnorm\_contrasts\_GLbulkRNAseq.csv
  - visualization\_output\_table\_ERCCnorm\_GLbulkRNAseq.csv
  - visualization\_PCA\_table\_ERCCnorm\_GLbulkRNAseq.csv

- Added parallel rRNA-removed DGE analysis:
  - Create filtered RSEM count files with rRNA features removed:
    - {sample}\_rRNA\_removed.genes.results

  - Normalize rRNA-removed counts
  - Perform DGE analysis using rRNA-removed counts
  - Output additional set of rRNA-removed counts and DGE results

A big thank you to @alexis.torres and @crystal.han on the Data Processing Team for leading this effort!

[@MicrobesAWG](https://awg.osdr.space/groups/microbesawg) [@AIMLawg](https://awg.osdr.space/groups/aimlawg) [@AnimalAWG](https://awg.osdr.space/groups/animalawg) [@ALSDAawg](https://awg.osdr.space/groups/alsdaawg) [@PlantAWG](https://awg.osdr.space/groups/plantawg) [@MultiOmicsAWG](https://awg.osdr.space/groups/multiomicsawg) [@HUMANawg](https://awg.osdr.space/groups/humanawg) [@RLWG](https://awg.osdr.space/groups/rlwg) [@FemaleReproAWG](https://awg.osdr.space/groups/femalereproawg)

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<div class="post-metadata">

**Author:** ![asaravia](https://sea1.discourse-cdn.com/flex001/user_avatar/awg.osdr.space/asaravia/32/290_2.png) [@asaravia](https://awg.osdr.space/u/asaravia)\
**Post date:** [February 12, 2025, 6:32pm UTC](https://awg.osdr.space/t/updated-rnaseq-pipeline-awg-review-needed/1780/2 "2025-02-12T18:32:48Z")

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Hi all,  
Just posting a reminder to please review the Eukaryotic bulk RNAseq pipeline updates and let me know if you have any issues, questions, or concerns by **Monday 2/17/2025**.