Mismatch in Read Counts Between *_read_aln_stats.tsv and per-file-stats.tsv

[LucileSol]

Operating System

Other Linux (please specify below)

Other Linux

Ubuntu 4.15.0-213-generic

Workflow Version

v1.6.1

Workflow Execution

Command line (Cluster)

Other workflow execution

No response

EPI2ME Version

No response

CLI command run

nextflow run wf-transcriptomes --fastq 'Retina_fix.fastq.gz' --direct_rna 'True' --out_dir 'Retina' --ref_genome 'genome_chr.fasta' --igv 'True' -profile singularity

Workflow Execution - CLI Execution Profile

singularity

What happened?

In my output folder I am looking at the statistics.

if I run

cat Retina_fix_read_aln_stats.tsv 

I see

sample_id PrimAlnPerc MultimapPerc PrimAln SecAln SupAln Unmapped TotalReads TotalRecords
Retina_fix 100.00 0.00 3047922 0 0 0 3047922 3047922

Here I can see 3047922 reads with 0 unmapped

If I run :

cat fastq_ingress_results/Retina_fix/fastcat_stats/per-file-stats.tsv 

I see :

filename        sample_name     n_seqs  n_bases min_length      max_length      mean_quality
input_src       Retina_fix      3357099 4011748863      50      16102   14.46

I have 3357099 reads and I know this is my input.

So what is the number 3047922 is it the number of reads mapped and then 0 is incorrect and it should be then 309177 Unmapped.

I would like to know where did those reads go and if they are just unmapped then it should be reported properly.

Thank you!

Relevant log output

N E X T F L O W   ~  version 24.10.5

Launching `/home/XXX/git/wf-transcriptomes/main.nf` [festering_keller] DSL2 - revision: 8620f10515


||||||||||   _____ ____ ___ ____  __  __ _____      _       _
||||||||||  | ____|  _ \_ _|___ \|  \/  | ____|    | | __ _| |__  ___
|||||       |  _| | |_) | |  __) | |\/| |  _| _____| |/ _` | '_ \/ __|
|||||       | |___|  __/| | / __/| |  | | |__|_____| | (_| | |_) \__ \
||||||||||  |_____|_|  |___|_____|_|  |_|_____|    |_|\__,_|_.__/|___/
||||||||||  wf-transcriptomes v1.6.1
--------------------------------------------------------------------------------
Core Nextflow options
  runName        : festering_keller
  containerEngine: singularity
  container      : [withLabel:isoforms:ontresearch/wf-transcriptomes:shad8671ea3a8ed52f2c0f40355e8eb5c6f00d2cbda, withLabel:wf_common:ontresearch/wf-common:shaabceef445fb63214073cbf5836fdd33c04be4ac7]
  launchDir      : /projects/annotation/XXX/RNAseq/ONT_analyses
  workDir        : /projects/annotation/XXX/RNAseq/ONT_analyses/work
  projectDir     : /home/XXX/git/wf-transcriptomes
  userName       : XXX
  profile        : singularity
  configFiles    : /home/XXX/git/wf-transcriptomes/nextflow.config

Input Options
  fastq          : /projects/annotation/XXX/qc/selection/Retina_fix.fastq.gz
  ref_genome     : /projects/annotation/XXX/qc/selection/genome_chr.fasta
  direct_rna     : true

Output Options
  out_dir        : Retina
  igv            : true

Advanced Options
  threads        : 8

!! Only displaying parameters that differ from the pipeline defaults !!
--------------------------------------------------------------------------------
If you use epi2me-labs/wf-transcriptomes for your analysis please cite:

* The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x


--------------------------------------------------------------------------------
This is epi2me-labs/wf-transcriptomes v1.6.1.
--------------------------------------------------------------------------------
Warning: As no --ref_annotation was provided, the output transcripts will not be annotated.
Searching input for [.fastq, .fastq.gz, .fq, .fq.gz] files.
[-        ] fastcat                        - 

...

Waiting for file transfers to complete (2 files)
Completed at: 20-Mar-2025 10:57:14
Duration    : 1h 13m 29s
CPU hours   : 9.3
Succeeded   : 77

Application activity log entry

Were you able to successfully run the latest version of the workflow with the demo data?

yes

Other demo data information

[sarahjeeeze]

Hi, I agree its a bit odd, Retina_fix_read_aln_stats.tsv is just counts of primary alignments which are used downstream in the analysis as we filtered for primary before getting this stats file. Where as the stats one is all the reads.

[LucileSol]

Thank you for your answer!
I did count the reads in my bam output and I have 3047922 reads in it.
Then I guess, it is a bug in the script that does the stats, and it does not count properly the unmapped reads (or does not report it properly).

[sparthib]

Hi @sarahjeeeze, I have a follow up qsn on this. How are these two files related to the table that has the number of secondary and supplementary alignments per sample in the html report?

For example: Here's my seqkit report:

The number of primary alignments here is different from the primary alignments per sample reported under the differential expression section.

Thanks!
Sowmya