Mapping of RNAseq reads.

Reads were mapped to the Ancylostoma ceylanicum genome described by in Schwartz et al 2015 and downloaded from WormBase ParaSite version WBPS14. Mapping of reads was performed with STAR version=2.7.5a

First, the genome was indexed:

STAR --runMode genomeGenerate --genomeDir star_index_wbps14/ --genomeFastaFiles ancylostoma_ceylanicum.PRJNA231479.WBPS14.genomic.fa

Note that the directory used for destination of index files needs to be made beforehand.

For reads mapping, the general command is as shown below using sample SRR6359160 asn example.

STAR --runMode alignReads --outFileNamePrefix SRR6359160 --outSAMtype BAM SortedByCoordinate --runMode alignReads --outFilterMultimapNmax 1000 --outSAMmultNmax 1 --outFilterMismatchNmax 3 --outMultimapperOrder Random --winAnchorMultimapNmax 1000 --alignEndsType EndToEnd --alignIntronMax 1 --alignMatesGapMax 350 --genomeDir --readFilesIn SRR6359160_1.fastq.gz SRR6359160_2.fastq.gz

The output files will be written to a directory called “SRR6359160” and the file with name “Aligned.sortedByCoord.out.bam” has the mapped reads.

We than changed all the file names so they contain the sample accession. For instance:

mv SRR6359160/Aligned.sortedByCoord.out.bam SRR6359160/SRR6359160_star.bam

These files were used as input for featurCounts v2.0.1.

featureCounts -M -F GFF -s 0 -p -t exon -g gene_id -o acey_avp25_annot_star_pe_fc.o -a acey_avp25_annot.gff SRR6359160/SRR6359160_star.bam SRR6359161/SRR6359161_star.bam SRR6359162/SRR6359162_star.bam ...

The three dots at the end of the command above represent additional BAM files that can be added. Featurecounts takes all BAM files in one single command and returns a counts table with gene_ids as rows and samples as columns.

Annotation file

The GFF file containing the annotation has been modified from the original annotation file available from Wormbase ParaSite. In brief, we added a gene_id field to the 9th column of the GFF so the read summary could be done by gene_id, as this level feature did not appear in the original annotation.

Im detail, the annotation file was obtained from Wormbase Parasite:

wget ftp://ftp.ebi.ac.uk/pub/databases/wormbase/parasite/releases/WBPS14/species/ancylostoma_ceylanicum/PRJNA231479/ancylostoma_ceylanicum.PRJNA231479.WBPS14.annotations.gff3.gz

unzipped:

gunzip ancylostoma_ceylanicum.PRJNA231479.WBPS14.annotations.gff3.gz

and the following script was used to modifiy the 9th column and write to a new file:

perl -lne 'my @line=split /\s+/; if ($line[8]=~m/Parent=transcript:(Acey_s\d+\.\w+)\.t\d/) {print $_.";gene_id=".$1};' < ancylostoma_ceylanicum.PRJNA231479.WBPS14.annotations.gff3 > acey_avp25_annot.gff

The modified annotation file has been submitted as supplementary materials with this manuscript.