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# Sample output files for tximport

This data package provides a set of output files from running a number
of various transcript abundance quantifiers on 2 samples from the
[GEUVADIS Project](http://www.geuvadis.org/web/geuvadis) (6 samples
for Cufflinks), as well as a number of other datasets described
below. The files are contained in the `inst/extdata` directory.

A citation for the GEUVADIS Project is:

> Lappalainen, et al., "Transcriptome and genome sequencing uncovers
> functional variation in humans", Nature 501, 506-511 (26 September
> 2013) [doi:10.1038/nature12531](http://dx.doi.org/10.1038/nature12531).

The purpose of this vignette is to detail which versions of software
were run, and exactly what calls were made.

# Inferential replicate data moved to Zenodo

Previous versions of this package included Salmon Gibbs samples
(`salmon_gibbs`) and kallisto bootstrap samples (`kallisto_boot`), as
well as all 6 GEUVADIS samples for each quantifier, the GENCODE v48
GTF file, and other larger files. To reduce the size of the package,
these were removed in version 1.41.1. The full set of files is
archived on Zenodo:

<https://doi.org/10.5281/zenodo.22982575>

# Sample information and quantification files

A small file, `samples.txt` is included in the `inst/extdata` directory:

```{r}
dir <- system.file("extdata", package="tximportData")
samples <- read.table(file.path(dir,"samples.txt"), header=TRUE)
samples
```

Further details can be found in a more extended table:

```{r}
samples.ext <- read.delim(file.path(dir,"samples_extended.txt"), header=TRUE)
colnames(samples.ext)
```

The quantification outputs themselves can be found in sub-directories:

```{r}
list.files(dir)
list.files(file.path(dir,"cufflinks"))
list.files(file.path(dir,"rsem","ERR188021"))
list.files(file.path(dir,"kallisto","ERR188021"))
list.files(file.path(dir,"salmon","ERR188021"))
list.files(file.path(dir,"sailfish","ERR188021"))
list.files(file.path(dir,"alevin"))
```

# Genome and gene annotation file

* For Cufflinks and Sailfish, the Illumina iGenomes was used as the index, see details below. 
* For RSEM, Salmon and kallisto, 
  the Gencode v27 CHR transcripts were used (`gencode.v27.transcripts.fa`).
* For the `salmon_dm` directory, the Ensembl Drosophila melanogaster
  v92 transcripts were used (either just cDNA or combining cDNA with
  non-coding transcripts).

Illumina iGenomes: The human genome and annotations were downloaded from
[Illumina iGenomes](https://support.illumina.com/sequencing/sequencing_software/igenome.html)
for the UCSC hg19 version. The human genome FASTA file used was in the
`Sequence/WholeGenomeFasta` directory and the gene annotation GTF file used
was the `genes.gtf` file in the `Annotation/Genes` directory. This GTF
file contains RefSeq transcript IDs and UCSC gene names. The
`Annotation` directory contained a `README.txt` file with the text:

> The contents of the annotation directories were downloaded from UCSC
> on: June 02, 2014.

The `genes.gtf` file was filtered to include only chromosomes
1-22, X, Y, and M.

# Cufflinks

Tophat2 version 2.0.11 was run with the call:

```
tophat -p 20 -o tophat_out/$f genome fastq/$f\_1.fastq.gz fastq/$f\_2.fastq.gz;
```

Cufflinks version 2.2.1 was run with the call:

```
cuffquant -p 40 -b $GENO -o cufflinks/$f genes.gtf tophat_out/$f/accepted_hits.bam;
```

Cuffnorm was run on all 6 GEUVADIS samples with the call:

```
cuffnorm genes.gtf -o cufflinks/ \
cufflinks/ERR188297/abundances.cxb \
cufflinks/ERR188088/abundances.cxb \
cufflinks/ERR188329/abundances.cxb \
cufflinks/ERR188288/abundances.cxb \
cufflinks/ERR188021/abundances.cxb \
cufflinks/ERR188356/abundances.cxb 
```

# RSEM

RSEM version 1.2.31 was run with the call:

```
rsem-calculate-expression --num-threads 6 --bowtie2 --paired-end <(zcat fastq/$f\_1.fastq.gz) <(zcat fastq/$f\_2.fastq.gz) index rsem/$f/$f
```

# kallisto

kallisto version 0.43.1 was run with the call:

```
kallisto quant --bias -i index -t 6 -o kallisto/$f fastq/$f\_1.fastq.gz fastq/$f\_2.fastq.gz
```

Only the `abundance.tsv.gz` files are included (these runs have no
bootstrap samples).

# Salmon

Salmon version 0.8.2 was run with the call:

```
salmon quant -p 6 --gcBias -i index -l IU -1 fastq/$f\_1.fastq.gz -2 fastq/$f\_2.fastq.gz -o salmon/$f
```

For the files in the `salmon_dm` directory (Drosophila melanogaster),
Salmon version 0.10.2 was run (once with only cDNA, once combining
cDNA with non-coding transcripts):

```
salmon quant -l A --gcBias --seqBias --posBias -i Drosophila_melanogaster.BDGP6.cdna.v92_salmon_0.10.2 -o SRR1197474 -1 SRR1197474_1.fastq.gz -2 SRR1197474_2.fastq.gz
```

For the files in the `salmon_ec` directory, 
Salmon version 1.1.0 was run with `--dumpEq` on the files from
Tasic, B., Yao, Z., Graybuck, L.T. *et al.* 
"Shared and distinct transcriptomic cell types across neocortical
areas" (2018)
[doi: 10.1038/s41586-018-0654-5](https://doi.org/10.1038/s41586-018-0654-5)
These files were generated by Jeroen Gilis. The raw data is from:
<https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA476008&o=acc_s%3Aa>

# alevin

Two small examples of `alevin` output (50 cells each) were generated by
Jeroen Gilis. The dataset is a subset from the paper, 
Hagai *et al.* "Gene expression variability across cells and species
shapes innate immunity" (2018) 
[doi: 10.1038/s41586-018-0657-2](https://doi.org/10.1038/s41586-018-0657-2)
Salmon/alevin version 1.6.0 was run, and using the tx2gene data that
is included in the package under `tx2gene_alevin.tsv`.

# oarfish

Three samples were processed with `oarfish` v0.9.0, downloaded from the 
[fastq download page](http://sg-nex-data.s3-website-ap-southeast-1.amazonaws.com/#data/sequencing_data_ont/fastq/) of the [SG-NEx Project](https://registry.opendata.aws/sgnex/). See also this [GitHub page](https://github.com/GoekeLab/sg-nex-data).

The samples were `SGNex_H9_cDNA` replicates 2-4, run 4.

These three samples were quantified with `oarfish` against human GENCODE v48 supplemented with "novel" sequences of length 1,000 bp across chr1-22 (500 per chromosome = 11,000 new transcripts named `novel1`, `novel2`, ...).

Code for the quantification steps are posted to a 
[GitHub repo](https://github.com/mikelove/oarfish4tximportData).

Briefly the following lines were used:

```
oarfish --only-index --annotated gencode.v48.transcripts.fa.gz --novel novel.fa.gz --seq-tech ont-cdna --threads 32 --index-out gencode_plus_novel
oarfish --reads reads/SGNex_H9_cDNA_replicateXYZ_run4.fastq.gz --index gencode_plus_novel --output quants/sgnex_h9_rep2 --seq-tech ont-cdna --filter-group no-filters --threads 32
```

Citation for the SG-NEx Project:

> Chen, Y., Davidson, N.M., Wan, Y.K. et al. A systematic benchmark of Nanopore long-read RNA sequencing for transcript-level analysis in human cell lines. Nat Methods 22, 801–812 (2025). <https://doi.org/10.1038/s41592-025-02623-4>

# Sailfish

Sailfish version 0.9.0 was run with the call:

```
sailfish quant -p 10 --biasCorrect -i sailfish_0.9.0/index -l IU -1 <(zcat fastq/$f\_1.fastq.gz) -2 <(zcat fastq/$f\_2.fastq.gz) -o sailfish_0.9.0/$f
```

# Session info

```{r}
sessionInfo()
```