> For the complete documentation index, see [llms.txt](https://ycl6.gitbook.io/rna-seq-data-analysis/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://ycl6.gitbook.io/rna-seq-data-analysis/de_novo_assembly_using_trinity.md).

# De novo assembly using Trinity

Trinity is one of the most popular software package for efficient and robust *de novo* reconstruction of transcriptomes from RNA-Seq data. It consists of three software modules, Inchworm, Chrysalis and Butterfly, that run sequentially to process the sequencing reads.

> Quote from [Trinity](https://github.com/trinityrnaseq/trinityrnaseq) GitHub:
>
> * **Inchworm** assembles the RNA-seq data into the unique sequences of transcripts, often generating full-length transcripts for a dominant isoform, but then reports just the unique portions of alternatively spliced transcripts.
> * **Chrysalis** clusters the Inchworm contigs into clusters and constructs complete de Bruijn graphs for each cluster. Each cluster represents the full transcriptional complexity for a given gene (or sets of genes that share sequences in common). Chrysalis then partitions the full read set among these disjoint graphs.
> * **Butterfly** then processes the individual graphs in parallel, tracing the paths that reads and pairs of reads take within the graph, ultimately reporting full-length transcripts for alternatively spliced isoforms, and teasing apart transcripts that corresponds to paralogous genes.

## Materials

The Trinity developers have provided [training materials](https://github.com/trinityrnaseq/RNASeq_Trinity_Tuxedo_Workshop/wiki), and the raw data and the software required are built into a VirtualBox image (Trinity2015.ova). I have saved a copy on **ALPS1**. The RNA-Seq data are 76 bp strand-specific Illumina RNA-Seq paired-end reads derived from *Schizosaccharomyces pombe* (fission yeast) grown under 4 conditions: 

1. logarithmic growth (Sp\_log)
2. plateau phase (Sp\_plat)
3. heat shock (Sp\_hs)
4. diauxic shift (Sp\_ds)

\* Due to the space limitation of gitbook, I will not provide the `fq.gz` files here, please obtain these files from the VirtualBox image \[[Link](https://data.broadinstitute.org/Trinity/RNASEQ_WORKSHOP/Trinity2015.ova)]

```
-rw-rw-r-- 1 ycl6 ycl6  5790168 Oct 27 11:35 RNASEQ_data/Sp_ds.left.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  5590326 Oct 27 11:35 RNASEQ_data/Sp_ds.right.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  5815390 Oct 27 11:35 RNASEQ_data/Sp_hs.left.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  5751383 Oct 27 11:36 RNASEQ_data/Sp_hs.right.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  2154125 Oct 27 11:36 RNASEQ_data/Sp_log.left.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  2097534 Oct 27 11:36 RNASEQ_data/Sp_log.right.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  5488286 Oct 27 11:36 RNASEQ_data/Sp_plat.left.fq.gz
-rw-rw-r-- 1 ycl6 ycl6  5238362 Oct 27 11:36 RNASEQ_data/Sp_plat.right.fq.gz
```

## Software

### [Trinity](https://github.com/trinityrnaseq/trinityrnaseq)

* **v2.2.0** \[17 Mar 2016] - Latest version available at the time of writing and used in this exercise
* v2.0.6 \[13 Mar 2015] - Latest version available on **ALPS1**

### [Bowtie](http://bowtie-bio.sourceforge.net/index.shtml)

* **v1.1.2** \[23 Jun 2015] - Latest version available at the time of writing and used in this exercise
* v1.0.1 \[14 Mar 2014] - Latest version available on **ALPS1**

### [GMAP](http://research-pub.gene.com/gmap) (Genomic Mapping and Alignment Program)

* **v2016-09-23** - Latest version available at the time of writing and used in this exercise

### [STAR](https://github.com/alexdobin/STAR) (Spliced Transcripts Alignment to a Reference)

* **v2.5.2b** \[20 Aug 2016] - Latest version available at the time of writing and used in this exercise
* v2.3.0e \[14 Feb 2013] - Latest version available on **ALPS1**

### [SAMtools](http://www.htslib.org/)

* **v1.3.1** \[22 Apr 2016] - Latest version available at the time of writing and used in this exercise
* v1.2 \[02 Feb 2015] - Latest version available on **ALPS1**

### [RSEM](https://github.com/deweylab/RSEM) (RNA-Seq by Expectation-Maximization)

* v1.3.0 \[02 Oct 2016] - Latest version available at the time of writing
* **v1.2.31** \[04 Jun 2016] - Version used in this exercise
* v1.2.19 \[05 Nov 2014] - Latest version available on **ALPS1**

## Set JAVA\_HOME and PATH

Bowtie 1 (*NOT* Bowtie 2) is required by the **Chrysalis** module.

\* Below is an example showing how to set up the paths, please remember to change the paths to these binaries accordingly.

```
cd ~/

export JAVA_HOME=/pkg/java/jdk1.7.0_51/bin/java

export PATH=/pkg/java/jdk1.7.0_51/bin:/pkg/biology/Bowtie/bowtie-1.0.1:\
/work3/LSLNGS2015/Tools/RSEM-1.2.23:/pkg/biology/R/R-3.1.2/bin:\
/work3/LSLNGS2015/Tools/gmap-2015-09-29/bin:/pkg/biology/samtools/samtools-1.2:\
/work3/LSLNGS2015/Tools/STAR-STAR_2.4.2a/bin/Linux_x86_64_static:\
/pkg/biology/trinity/trinityrnaseq-2.0.6:$PATH
```

You can use `echo $PATH` to check the new PATH variable.