Long non-coding RNA discovery in Pocillopora verrucosa

Project Background and Goal

  • Long non-coding RNAs (lncRNAs) are a type of RNA molecule that are longer than 200 nucleotides and do not encode for proteins.

  • Play important roles in various cellular processes

    • gene expression regulation

    • chromatin remodeling

    • post-transcriptional processing

Project Background and Goals

  • RNA-seq data from corals

    • Treatment: nutrient exposure

  • Identify long non-coding RNAs (lncRNAs) in RNA-seq data

    • Develop a list of all lncRNAs found in each sample for use in differential and/or correlation expression analysis

RNA-seq Data

  • Trimmed fastq files
@GWNJ-0957:702:GW2012083569th:7:1101:27722:1485 1:N:0:NCCGGAGA+NTTCGCCT
NTTTCTCTGAACACTTGCTTTTCTTCTTGTCAATCTGCAATAAATTGTAGAACATTATCAAATAAATAGGTTTGCTGAAAATAATTAAAATTAAAAGAGAATGAGTTGAGCTTTTTCTCTGTGTTATATATTTGAGATTGGAAGAGCACA
+
#A<A-FFFJJJAFA-FJJJJJJJJJJJJFFFJ<JFFFJJ7FJJJJJ--FJFFJJ-FJJ-FJFF<F-JJJF--<F-<FAFFJ--AFFFJJFJJJA<---A-7---<-FFFAJAFJJJ--AFA-FAJAJFJF<JJJJFJJ-7FAFJFJ--7F
@GWNJ-0957:702:GW2012083569th:7:1101:18619:1502 1:N:0:NCCGGAGA+NTTCGCCT
CGCAGTTCCTTAATATGGTGGATTACCATGATCAGTTGCACTCACTTGAAACCTTGACGGTGAAGGTGTTTCACGATCTAAAAGGCTCAACCCAGTAATCTTGCCAGTTTTAGAATTGATTTTAAAGTTTTGCAGAGAATGTTCAATTGC
+
AAFFFFFFJFF<F-AAFJJJJJJJJJJJJJJFFFJJJJJFJF-F7JJJFJJJJJJJFJA-JFJJ-<<JJJJJJJJJFJJJ<-F<AJFJJJJJJFAFJJJF-AFJ7<JF7JJFFJJJJJJJJ<JJJJJJJJAFJJJJJJFAFJJ7JJAJJ<
@GWNJ-0957:702:GW2012083569th:7:1101:20750:1502 1:N:0:NCCGGAGA+NTTCGCCT
CATCTGACAGGAGTGCTGTAGTACCACCTGTACATCCACAGTGAATTGCTGTCATCGTAGTGGTTAATTGATCTATACTGTGCTGCATCTTCTACAAACACTGCTGCCTGGTTAATTGCATGTCCTGATATATCATCGACATTAATTAAA

Methods: Workflow

Highlight: HISAT2

  • Run HISAT2 over all files to align
find /home/shared/8TB_HDD_01/pver/*gz \
| xargs basename -s _R1_001.fastq.gz | xargs -I{} \
/home/shared/hisat2-2.2.1/hisat2 \
-x ../output/Pver_genome_assembly_v1.0-valid.index \
-p 8 \
-1 /home/shared/8TB_HDD_01/pver/{}_R1_001.fastq.gz \
-2 /home/shared/8TB_HDD_01/pver/{}_R2_001.fastq.gz \
-S /home/shared/8TB_HDD_01/pver/hisat-output/{}-valid.sam

Highlight: CPC2

eval "$(/opt/anaconda/anaconda3/bin/conda shell.bash hook)"

python /home/shared/CPC2_standalone-1.0.1/bin/CPC2.py 
-i /home/shared/8TB_HDD_01/pver/bedtools-output/merged_lncRNA_candidates.fasta 
-o /home/shared/8TB_HDD_01/pver/cpc2-output/cpc2_results.txt

Current Place

awk 'NR>1 && $9 < 0 {print $1}' /home/shared/8TB_HDD_01/pver/cpc2-output
/cpc2_results.txt.txt > /home/shared/8TB_HDD_01/pver/cpc2-output
/noncoding_transcripts_ids.txt
grep -Fwf /home/shared/8TB_HDD_01/pver/cpc2-output/noncoding_transcripts_ids.txt 
/home/shared/8TB_HDD_01/pver/bedtools-output/merged_lncRNA_candidates.fasta > ~ 
  /github/zach-lncRNA/output/merged_final_lncRNAs.gtf
  • List of lncRNA transcript IDs for all samples
>transcript::Pver_Sc0000000_size2095917:2732-3551
>transcript::Pver_Sc0000000_size2095917:2850-3525
>transcript::Pver_Sc0000000_size2095917:21743-24773
>transcript::Pver_Sc0000000_size2095917:21768-22840
>transcript::Pver_Sc0000000_size2095917:21802-24097
>transcript::Pver_Sc0000000_size2095917:21849-23116
>transcript::Pver_Sc0000000_size2095917:21879-22840
>transcript::Pver_Sc0000000_size2095917:21880-23235
>transcript::Pver_Sc0000000_size2095917:21880-24857
>transcript::Pver_Sc0000000_size2095917:21882-22840

Where I’m headed…

Week 6 Kallisto count matrices
Week 7-8 Co-expression with coding RNAs
Weeks 9-10 DESEq2, lncRNA sensitivity to treatment