# Transcriptome Assembly – Geoduck RNAseq data

Used all of our current geoduck RNAseq data to assemble a transcriptome using Trinity.

Trinity was run our our Mox HPC node. Specifically, I had to use just a single node with 500GB of RAM. Trinity could not run with much less than that. Initially, I attempted to run with two nodes, but our smaller node (120GB) ended up limiting the available RAM (the system only uses the RAM available on the smallest node; it cannot combine RAM or dynamically allocate computing to a node with larger RAM when needed) and Trinity consistently crashed due to memory limitations.

Reads were trimmed using the built-in version of Trimmomatic with the default settings.

SBATCH script:

Due to the huge number of input files, I won’t post the entire script contents here. Instead, here’s a snippet of the script showing the commands used to start the Trinity run:


#!/bin/bash
## Job Name
#SBATCH --job-name=20180829_trinity
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=30-00:00:00
## Memory per node
#SBATCH --mem=500G
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --workdir=/gscratch/scrubbed/samwhite/20180827_trinity_geoduck_RNAseq

# Load Python Mox module for Python module availability

# Document programs in PATH (primarily for program version ID)

date >> system_path.log
echo "" >> system_path.log
echo "System PATH for $SLURM_JOB_ID" >> system_path.log echo "" >> system_path.log printf "%0.s-" {1..10} >> system_path.log echo${PATH} | tr : \n >> system_path.log

# Run Trinity
/gscratch/srlab/programs/trinityrnaseq-Trinity-v2.8.3/Trinity
--trimmomatic
--seqType fq
--max_memory 500G
--CPU 28


Despite the naming conventions, this job was submitted to the Mox scheduler on 20180829 and finished on 20180901.

After job completion, the entire folder was gzipped (the following method of gzipping is SUPER fast, btw):

tar -c 20180827_trinity_geoduck_RNAseq | pigz > 20180827_trinity_geoduck_RNAseq.tar.gz

#### RESULTS:

Output folder:

Trinity assembly (FastA):

Next up, I’ll get some annotations going by running through TransDecoder and blastx.

# Bioinformatics – Trimmomatic/FASTQC on C.gigas Larvae OA NGS Data

Previously trimmed the first 39 bases of sequence from reads from the BS-Seq data in an attempt to improve our ability to map the reads back to the C.gigas genome. However, Mac (and Steven) noticed that the last ~10 bases of all the reads showed a steady increase in the %G, suggesting some sort of bias (maybe adaptor??):

Although I didn’t mention this previously, the figure above also shows an odd “waves” pattern that repeats in all bases except for G. Not sure what to think of that…

Quick summary of actions taken (specifics are available in Jupyter notebook below):

• Trim first 39 bases from all reads in all raw sequencing files.
• Trim last 10 bases from all reads in raw sequencing files
• Concatenate the two sets of reads (400ppm and 1000ppm treatments) into single FASTQ files for Steven to work with.

Raw sequencing files:

Notebook Viewer: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC

Jupyter (IPython) notebook: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC.ipynb

### Output files

Trimmed, concatenated FASTQ files
20150521_trimmed_2212_lane2_400ppm_GCCAAT.fastq.gz
20150521_trimmed_2212_lane2_1000ppm_CTTGTA.fastq.gz

Example of FASTQC analysis pre-trim:

Example FASTQC post-trim (from 400ppm data):

Trimming has removed the intended bad stuff (inconsistent sequence in the first 39 bases and rise in %G in the last 10 bases). Sequences are ready for further analysis for Steven.

However, we still see the “waves” pattern with the T, A and C. Additionally, we still don’t know what caused the weird inconsistencies, nor what sequence is contained therein that might be leading to that. Will contact the sequencing facility to see if they have any insight.

# Bioinformatics – Trimmomatic/FASTQC on C.gigas Larvae OA NGS Data

In another troubleshooting attempt for this problematic BS-seq Illumina data, I’m going to use Trimmomatic to remove the first 39 bases of each read. This is due to the fact that even after the previous quality trimming with Trimmomatic, the first 39 bases still showed inconsistent quality:

Ran Trimmomatic on just a single data set to try things out: 2212_lane2_CTTGTA_L002_R1_001.fastq.gz

Notebook Viewer: 20150506_Cgigas_larvae_OA_trimmomatic_FASTQC

Jupyter (IPython) notebook: 20150506_Cgigas_larvae_OA_trimmomatic_FASTQC.ipynb

Results:

Trimmed FASTQ: 20150506_trimmed_2212_lane2_CTTGTA_L002_R1_001.fastq.gz

You can see how flat the newly trimmed data is (which is what one would expect).

Steven will take this trimmed dataset and try additional mapping with it to see if removal of the first 39 bases will improve the mapping.

# Quality Trimming – C.gigas Larvae OA BS-Seq Data

Jupyter (IPython) Notebook: 20150414_C_gigas_Larvae_OA_Trimmomatic_FASTQC.ipynb

# Quality Trimming – LSU C.virginica Oil Spill MBD BS-Seq Data

Jupyter (IPython) Notebook: 20150414_C_virginica_LSU_Oil_Spill_Trimmomatic_FASTQC.ipynb

### Trimmed FASTQC

#### HB30 25,000ppm oil Index – TGACCA

20150414_trimmed_2112_lane1_TGACCA_L001_R1_001_fastqc.html