Experimental Design:

• 4 populations of Fundulus grandis were collected across the Gulf of Mexico:

• Reference (clean) sites: Leeville, LA (LA-REF 29°15'24.6"N 90°12'51.3"W) and Gangs Bayou, Galveston, TX (TX-REF; 29°15'30.31"N; 95°54'45"W)

• Chemically Polluted sites: Grand Terre Island, LA, contaminated by the Deepwater Horizon oil spill (LA-DWH; 29° 16' 22.4472'' N 89° 56' 41.46'' W) and polluted Superfund site at Vince Bayou, Houston, TX (TX-SF; 29°43'10"N; 95°13'13"W)

• Fish from all four populations were held in common clean laboratory conditions (12 g/L artificial sea water [ASW] made with Instant Ocean salt mix; United Pet Group, Cleveland, OH, USA) for at least 2 generations at Louisiana State University prior to experiments. Adult fish were used for two purposes. Some adults were directly exposed to oil to test for exposure effects on reproductive fitness. Other adults were used as brood stock to create embryos that were used to test for oil exposure impacts on early-life development.

• Parent treatment: Adult fish were exposed to WAF or clean/control water for ~60 days before spawning

• Embryo exposure dosage: Embryos were exposed to WAF 00%, 10%, 32%, or 56% for 21 days

• Embryo developmental stage: Embryos were sampled for RNA-seq at stages 19, 28, 35, or hatch

  • Leftover embryos that were unhatched at the end of 21 days were collected as non-hatched (NH)

• RNA-seq data for all four populations:

  • ARS: Aquatic Research Station, aquacultured pop in LA
  • GT: Grand Terre, recently oiled pop in LA
  • VB: Vince Bayou, industrial pollution pop from Houston Ship Channel
  • GB: Gang's Bayou, clean ref pop near Houston Ship Channel

• Up to 5 replicates were sequenced for each treatment

• File naming convention:

  • [SampleNumber, 4 digits] ARS [ParentTreatment, 1 letter] [Embryo exposure %, 2 digits] [Developmental Stage, 2 digits] [ReplicateNumber 1-5]
  • Sample Number: unique number assigned to each embryo sequenced
  • ARS: population of F. grandis
  • Parent Treatment: Exposed (E) or Control (C)
  • Embryo exposure: 0, 10, 32, or 56%
  • Developmental Stage: 19, 28, 35, or HA
  • Replicate Number: Single digit, 1-5

• Samples 75, 145, 146, and 163 should be excluded from analysis due to barcoding error (see "Identifying samples that didn't get sequenced in the first lane trial")

General Pipeline:

• [Quality control with FastQC]
• [Trim short reads with Trimmomatic]
• [Map and quantify reads with Salmon]
• [Differential expression analysis in R]

Quality control with FastQC

FastQC is a raw read quality assessment command line tool. I used the following parameters to QC reads before and after trimming:

fastqc file --noextract -o ToDir		

Trim short reads with trimmomatic

Reads were trimmed lightly using Trimmomatic command line tool and NEBNext adaptor .fa file.

[0002_trimmomaticNEB.sh] link to the script

The .fa files are located at:

~/programs/Trimmomatic-0.36/adapters

Merge trimmed reads

Notes: looks like I copied the trimmed reads into intermediate mergedfq directories before merging and dumping into OUTDIR=~/niehs/Data/trimmed_data/mergedfq

Map and quantify reads with Salmon

Reference transcriptome:

• Lisa Cohen concatenated two [reference transcriptomes by Don Gilbert] (http://arthropods.eugenes.org/EvidentialGene/killifish/genes/kfish2rae5/) that contain alternate gene model information. I used that concatenation, found here:

    ~/niehs/refseq/kfish2rae5g.mrna.combined
  

• Trimmed and merged data located:

    ~/niehs/Data/trimmed_data/mergedfq/
  

Reads were ultimately mapped to Don Gilbert's reference transcriptome, fmd mode.

Differential Expression Analysis in R

Gene-level counts from transcript abundances were estimated using tximport in R.