B.tabaci's salivary glands genes identification

This repository includes all the codes, results and explanations for the identification of the differentially expressed cathepsin B and L genes from figures 5 and 6 in the article "Evolved transcriptional responses and their trade-offs after long-term adaptation of Bemisia tabaci to a marginally-suitable host". These genes are likely to be found at the salivary glands of the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) MEAM1.

The following files were obtained from the NCBI for the analysis:

1. RNAseq of salivary glands

   Identifiers: BioSample: SAMEA4682676; SRA: ERS2502869

   Organism: Bemisia tabaci

   Accession: SAMEA4682676 ID: 12215746

2. Identification of Saliva Proteins of the Whitefly Bemisia tabaci by Transcriptome

   Identifiers: BioSample: SAMN13381603 ; SRA: SRS5714533

   Organism: Bemisia tabaci

   Accession: SAMN13381603

All the steps are listed and described below.

General workflow:

1. File of 13 cathepsin B and L genes created.
2. Simmilrity of genes was accessed.
3. The genes blasted against 8,610 salivary gland genes in B.tabaci found in the NCBI.
4. Transcriptomic data from salivary glands of B.tabaci blasted in the NCBI online against the genes.
5. Transcriptomic data from salivary glands of B.tabaci was assembled using Trinity and genes were predicted using Transdecoder.
6. Transcriptomic data from salivary glands of B.tabaci was mapped against the genes.
7. The genes were analised for signal peptides (SP) in the webpage signalP.

Programs used:

UGENE, IGV, bowtie2, Trinity and Transdecoder.

Commands used for assembly and gene prediction from transcriptomic data

Commands used for BLASTing:

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makeblastdb -in Trinity_single.fasta -dbtype nucl -out Trinity_single.fasta

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makeblastdb -in Trinity_paired.fasta -dbtype nucl -out Trinity_paired.fasta

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for i in Trinity_paired.fasta Trinity_single.fasta; do blastn -query $i -db cathepsin.fa -out $i.results.txt -outfmt 6; done

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Results files can be found at the Results folder:

BLASTN_for_Trinity_paired.fasta.results.txt

BLASTN_for_Trinity_single.fasta.results.txt

Commands used for mapping:

The reads where mapped and manually analyzed using the following commands:

1. Indexing of the database ($i == Transcripts of cathepsin B and L genes):

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for i in XM_*.fa; do bowtie2-build $i $i; done

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2. Mapping of all the obtained reads to the refrence database created in step 1:

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for i in XM_*.fa; do bowtie2 --fast -p 16 -x $i -1 *_R1.fastq.gz -2 *_R2.fastq.gz -U Single_R.fastq.gz -S $i.sam

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3. Converting from SAM to BAM and indexing the BAM for data visualization in IGV:

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for i in XM_*.sam ; do samtools view -bS $i | samtools sort -o $i.sorted.bam ; done

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for i in XM_*.sorted.bam; do samtools index $i $i.bai; done

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The output was visualized in IGV and PNG of each gene with his covarge was exported.

The output of this analysis

Identification of the genes as genes from the salivary gland using trabscriptomic data

Blast against the reads using the NCBI platform and against the predicted genes from the reads were not sensitive enough.

Therfore we analysed the obtained reads from the NCBI mapping them against the 13 genes of cathepsin which resulted in:

1.Seven genes had high and equal coverage.

Gene	mRNA	Protein	Reads Coverage
LOC109031579	XM_019043171.1	XP_018898716.1
LOC109031589	XM_019043181.1	XP_018898726.1
LOC109032360	XM_019044441.1	XP_018899986.1
LOC109036811	XM_019051187.1	XP_018906732.1
LOC109039209	XM_019054602.1	XP_018910147.1
LOC109042507	XM_019059295.1	XP_018914840.1
LOC109042510	XM_019059300.1	XP_018914845.1

2. One of the references cathepsin genes had small amounts of reads mapping to it but equally distributed over all the gene.

Gene	mRNA	Protein	Reads Coverage
LOC109042327	XM_019059005.1	XP_018914550.1

3. Five genes were reads mapped poorly and inconsistent.

Gene	mRNA	Protein	Reads Coverage
LOC109031789	XM_019043541.1	XP_018899086.1
LOC109032723	XM_019044995.1	XP_018900540.1
LOC109032724	XM_019045001.1	XP_018900546.1
LOC109040923	XM_019057048	XP_018912593.1
LOC109042262	XM_019058909.1	XP_018914454.1

Identification of the genes as genes from the salivary gland using signal peptides

Gene	mRNA	Protein	Signal peptide analysis
LOC109031579	XM_019043171.1	XP_018898716.1
LOC109031589	XM_019043181.1	XP_018898726.1
LOC109031789	XM_019043541.1	XP_018899086.1
LOC109032360	XM_019044441.1	XP_018899986.1
LOC109032723	XM_019044995.1	XP_018900540.1
LOC109032724	XM_019045001.1	XP_018900546.1
LOC109036811	XM_019051187.1	XP_018906732.1
LOC109039209	XM_019054602.1	XP_018910147.1
LOC109040923	XM_019057048	XP_018912593.1
LOC109042262	XM_019058909.1	XP_018914454.1
LOC109042327	XM_019059005.1	XP_018914550.1
LOC109042507	XM_019059295.1	XP_018914840.1
LOC109042510	XM_019059300.1	XP_018914845.1

Results PDF for the analysis

Supplementary Data:

Genes simmilarity analysis: Information about the precentage of identity between the cathepsin B and L genes.

BLAST output file for step 3 in the general wokflow

BLAST output file for step 5 in the general wokflow

Who do I talk to?

• Repo members or admin