Pipeline used in:

Gene expression profiling across ontogenetic stages in the wood white (Leptidea sinapis) reveals pathways linked to butterfly diapause regulation

Luis Leal, Venkat Talla, Thomas Källman, Magne Friberg, Christer Wiklund, Vlad Dincă, Roger Vila, and Niclas Backström. Molecular Ecology 27 (4), 935–48 (2018). https://doi.org/10.1111/mec.14501.

---

Luis Leal, Uppsala University, Uppsala, Sweden, 2017

---

1. QUALITY CONTROL

• Filter libraries using TrimGalore
• Mask libraries using Fastq Masker
• Filter libraries using prinseq
• Filter libraries using condetri
• Screen libraries using fastQ Screen
• Reconcile paired-end libraries after using fastQ Screen (removes non-paired reads)

(Note: Index fasta files as required.)

01_quality_control.sh

---

2. DE NOVO TRANSCRIPTOME ASSEMBLY USING TRINITY

(Note: Index fasta files as required.)

02_trinity_transcriptome_assembly.sh

---

3. FUNCTIONAL ANNOTATION BASED ON TRINOTATE PROTOCOL

• a. Annotate each trinity transcript using modified Trinotate protocol

03_trinotate_functional_annotation.sh

• b. Build local UniProt databases

04_build_local_UniProt_db.sh

• c. Find GO terms associated to each Trinity gene

05_associate_GO_terms_to_genes.sh

---

4. TRANSCRIPT QUANTIFICATION USING KALLISTO

(Note: Index files as required.)

06_kallisto_transcript_quantification.sh

---

5. GENE COUNT CONSOLIDATION USING TXIMPORT

• a. Convert counts-per-transcript to counts-per-gene using tximport

  • a1. Associate Trinity transcript name to its gene name

  07_associate_transcript_to_gene.py

  • a2. Convert Kallisto output files from count-per-transcript to count-per-gene

  08_tximport_aggregate_transcript_counts.R

• b. Consolidate annotation file & gene counts

  09_tximport_consolidateGenes.py

---

6. DIFFERENTIAL GENE EXPRESSION ANALYSIS USING DESEQ2

10_DESeq2.R

---

7. GENE ONTOLOGY ENRICHMENT ANALYSIS USING TOPGO

• a. Create two-column file that lists each Trinity gene and its associated GO terms

11_gene_to_GO.py

• b. Perform gene ontology enrichment analysis

12_TopGO.R

---