### StringTie options

The following optional parameters can be specified (use `-h` or `--help` to get the usage message):

```
 --mix : both short and long read data alignments are provided
        (long read alignments must be the 2nd BAM/CRAM input file)
 --rf : assume stranded library fr-firststrand
 --fr : assume stranded library fr-secondstrand
 -G reference annotation to use for guiding the assembly process (GTF/GFF)
 --conservative : conservative transcript assembly, same as -t -c 1.5 -f 0.05
 --ptf : load point-features from a given 4 column feature file <f_tab>
 -o output path/file name for the assembled transcripts GTF (default: stdout)
 -l name prefix for output transcripts (default: STRG)
 -f minimum isoform fraction (default: 0.01)
 -L long reads processing; also enforces -s 1.5 -g 0 (default:false)
 -R if long reads are provided, just clean and collapse the reads but
    do not assemble
 -m minimum assembled transcript length (default: 200)
 -a minimum anchor length for junctions (default: 10)
 -j minimum junction coverage (default: 1)
 -t disable trimming of predicted transcripts based on coverage
    (default: coverage trimming is enabled)
 -c minimum reads per bp coverage to consider for multi-exon transcript
    (default: 1)
 -s minimum reads per bp coverage to consider for single-exon transcript
    (default: 4.75)
 -v verbose (log bundle processing details)
 -g maximum gap allowed between read mappings (default: 50)
 -M fraction of bundle allowed to be covered by multi-hit reads (default:1)
 -p number of threads (CPUs) to use (default: 1)
 -A gene abundance estimation output file
 -E define window around possibly erroneous splice sites from long reads to
    look out for correct splice sites (default: 25)
 -B enable output of Ballgown table files which will be created in the
    same directory as the output GTF (requires -G, -o recommended)
 -b enable output of Ballgown table files but these files will be 
    created under the directory path given as <dir_path>
 -e only estimate the abundance of given reference transcripts (requires -G)
 --viral : only relevant for long reads from viral data where splice sites
    do not follow consensus (default:false)
 -x do not assemble any transcripts on the given reference sequence(s)
 -u no multi-mapping correction (default: correction enabled)
 --ref/--cram-ref reference genome FASTA file for CRAM input

Transcript merge usage mode: 

  stringtie --merge [Options] { gtf_list | strg1.gtf ...}
With this option StringTie will assemble transcripts from multiple
input files generating a unified non-redundant set of isoforms. In this mode
the following options are available:
  -G <guide_gff>   reference annotation to include in the merging (GTF/GFF3)
  -o <out_gtf>     output file name for the merged transcripts GTF
                    (default: stdout)
  -m <min_len>     minimum input transcript length to include in the merge
                    (default: 50)
  -c <min_cov>     minimum input transcript coverage to include in the merge
                    (default: 0)
  -F <min_fpkm>    minimum input transcript FPKM to include in the merge
                    (default: 1.0)
  -T <min_tpm>     minimum input transcript TPM to include in the merge
                    (default: 1.0)
  -f <min_iso>     minimum isoform fraction (default: 0.01)
  -g <gap_len>     gap between transcripts to merge together (default: 250)
  -i               keep merged transcripts with retained introns; by default
                   these are not kept unless there is strong evidence for them
  -l <label>       name prefix for output transcripts (default: MSTRG)

```

More details about StringTie options can be found in the [online manual](http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual).

## Input files

StringTie takes as input a SAM, BAM or CRAM file sorted by coordinate (genomic location). 
This file should contain spliced RNA-seq read alignments such as the ones produced by TopHat or HISAT2.
TopHat output is already sorted. Unsorted SAM or BAM files generated by other aligners should be sorted using the `samtools` program:
```
samtools sort -o alns.sorted.bam alns.sam
```
The file resulted from the above command (alns.sorted.bam) can be used as input to StringTie. 

Any SAM record with a spliced alignment (i.e. having a read alignment across at least one junction)
should have the `XS` tag to indicate the transcription strand, i.e. the genomic strand from which the RNA that produced
this read originated. TopHat and HISAT2 alignments already include this tag, but if you use
a different read mapper you should check that this tag is also included for spliced alignment
records. STAR aligner should be run with the option `--outSAMstrandField intronMotif` in order to generate this tag.

The `XS` tags are not necessary in the case of long RNA-seq reads aligned with `minimap2` using the `-ax splice` option. `minimap2` adds the `ts` tags to splice alignments to indicate the transcription strand (albeit in a different manner than the `XS` tag), and StringTie can recognize the `ts` tag as well, if the `XS` tag is missing. 
Thus the long read spliced alignments produced by `minimap2` can be also assembled by StringTie (with the option `-L` or 
as the 2nd input file for the `--mix` option).

As explained above, the alignments must be sorted by coordinate before they can be used as input for StringTie.

When CRAM files are used as input, the original reference genomic sequence can be provided with the `--ref` (`--cram-ref`) option 
as a multi-FASTA file with the same chromosome sequences that were used when aligning the reads. This is optional but recommended because StringTie
can better estimate the quality of some spliced alignments (e.g. noticing mismatches around junctions) and that data can be retrieved 
in the case of some CRAM files only when the reference genome sequence is also provided. 

### Reference transcripts (guides)

A reference annotation file in GTF or GFF3 format can be provided to StringTie 
using the `-G` option which can be used as 'guides' for the assembly process. 

When the `-e` option is used (i.e. expression estimation only), this option is required, 
and in that case StringTie will not attempt to assemble the read alignments but instead it will 
only estimate the expression levels of all the transcripts provided in this file

Note that when a reference transcript is fully covered by reads, the original transcript ID from the reference annotation file will be 
shown in StringTie's output record in the _`reference_id`_ GTF attribute. Output transcripts that lack such `reference_id` attribute 
can be considered "novel" transcript structures with respect to the given reference annotation.

## The super-reads module

This optional module can be used to de-novo assemble, align and pre-process
RNA-Seq reads, preparing them to be used as "super-reads" by Stringtie.

More usage information is provided in <a href="https://github.com/gpertea/stringtie/blob/master/SuperReads_RNA/README.md">SuperReads_RNA/README.md</a>.
Quick installation instructions for this module from the source available on this repository (assuming main Stringtie installation was already completed as described above):

```
 cd SuperReads_RNA
 ./install.sh
```

### Using super-reads with Stringtie

After running the super-reads module (see the <a href="https://github.com/gpertea/stringtie/blob/master/SuperReads_RNA/README.md">SuperReads_RNA</a> module documentation for usage details), there 
is a BAM file created which contains sorted alignment for both short reads and super-reads, called *`sr_merge.bam`*, 
created in the selected output directory. This file can be directly given as the main input file
to StringTie as described in the [Running StringTie](#running-stringtie) section above.


## License
StringTie is free, open source software released under an <a href="https://opensource.org/licenses/MIT">MIT License</a>.

## Publications
Shumate A, Wong B, Pertea G, Pertea M [**Improved transcriptome assembly using a hybrid of long and short reads with StringTie**](https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1009730), _PLOS Computational Biology_ 18, 6 (2022), doi.org/10.1371/journal.pcbi.1009730 

Kovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M  [**Transcriptome assembly from long-read RNA-seq alignments with StringTie2**](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1910-1), _Genome Biology_ 20, 278 (2019),  doi:10.1186/s13059-019-1910-1

Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL [**Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown**](http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html), _Nature Protocols_ 11, 1650-1667 (2016), doi:10.1038/nprot.2016.095

Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT  & Salzberg SL [**StringTie enables improved reconstruction of a transcriptome from RNA-seq reads**](http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3122.html), _Nature Biotechnology_ 2015, doi:10.1038/nbt.3122