# De novo assembly of RNA-Seq reads

## *De novo* assembly of reads

First, we combine the raw reads from different conditions into a single FASTQ file (for each end) and use Trinity to generate a **reference** assembly.

\* Assume the `fq.gz` files were already placed in the `Trinity/RNASEQ_data` folder

```
mkdir ~/LSLNGS2015/Trinity
mkdir ~/LSLNGS2015/Trinity/RNASEQ_data/
```

### Execute

```
cd ~/LSLNGS2015/Trinity

zcat RNASEQ_data/*.left.fq.gz | gzip > RNASEQ_data/sp.left.fq.gz
zcat RNASEQ_data/*.right.fq.gz | gzip > RNASEQ_data/sp.right.fq.gz

bsub -q 16G -o ./trinity_reference.std -e ./trinity_reference.err -J Trinity \
"Trinity --seqType fq --SS_lib_type RF \
--left RNASEQ_data/sp.left.fq.gz --right RNASEQ_data/sp.right.fq.gz \
--CPU 10 --max_memory 16G --output trinity_reference >& trinity_reference.log"
```

You will get a message like `Job <xxxxxx> is submitted to queue <16G>.` to let you know your submission is successful.

You can use `bjobs` to check your job status.

```
JOBID   USER    STAT  QUEUE      FROM_HOST   EXEC_HOST   JOB_NAME   SUBMIT_TIME
993732  s00ycm0 RUN   16G        alps1       8*alps1-40  Trinity    Nov 18 22:38
```

### Resource usage

| ALPS Queue Name | CPU Time     | Max Memory | Duration             |
| --------------- | ------------ | ---------- | -------------------- |
| 16G             | 2097.24 sec. | 2 GB       | 6 minutes 37 seconds |

The assembled transcripts can be found at `trinity_reference/Trinity.fasta`.

## Examine assembly stats

The `TrinityStats.pl` script will report the basic statistics about the assembly produced by Trinity. The numbers may vary slightly, as the assembly results are not deterministic.

### Execute

Locate `util/TrinityStats.pl` in the trinityrnaseq-2.2.0 distribution, and run

`PATH_TO_TRINITY/util/TrinityStats.pl trinity_reference/Trinity.fasta`

### Output

```
################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':  377
Total trinity transcripts:      385
Percent GC: 38.65

########################################
Stats based on ALL transcript contigs:
########################################

        Contig N10: 3373
        Contig N20: 2605
        Contig N30: 2219
        Contig N40: 1936
        Contig N50: 1703

        Median contig length: 772
        Average contig: 1046.43
        Total assembled bases: 402875


#####################################################
## Stats based on ONLY LONGEST ISOFORM per 'GENE':
#####################################################

        Contig N10: 3373
        Contig N20: 2605
        Contig N30: 2219
        Contig N40: 1936
        Contig N50: 1682

        Median contig length: 765
        Average contig: 1038.27
        Total assembled bases: 391428
```


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