Helminths

Prediction accuracy with parameters trained by WebAUGUSTUS and by human experts was measured using three different data sets. For optA, the genome of the insect Drosophila melanogaster (assembly BDGP R5/dm3) and 818 005 ESTs from the same species that were obtained from the National Center for Biotechnology Information (NCBI) were used. OptB was evaluated using the genome of the plant Arabidopsis thaliana (assembly TAIR 10) and 35 375 protein sequences of the same species that were obtained from NCBI. OptC was evaluated using the genome of the worm Caenorhabditis elegans and 18 555 training gene structures retrieved from Wormbase (16 (link)).
To avoid an overly optimistic performance estimate for the new genes, the chromosomes of all genomes [for fly and plant downloaded from the UCSC Genome Browser database (17 (link))] were split into two parts in such a way that ∼50% of the genes were located on the first half, and the remaining genes were located on the second half. The second part of all chromosomes was used as a genomic input sequence for training AUGUSTUS, whereas the first part served for accuracy assessment opf gene predictions.
For D.melanogaster, protein coding genes from FlyBase (18 (link)), for A. thaliana, protein coding genes from TAIR 10 (19 (link)) and for C. elegans, protein coding genes from Wormbase were used as a reference annotation for measuring accuracy.
The exact source of all data sets and the files used for the actual experiments are described in detail in Supplementary Materials, section Supplementary Methods: Data Sets.
Commonly used measures of accuracy (measured in percent) in gene prediction are

where TP stand for true positives, i.e. the number of predicted features that agree with the gold-standard reference, FN stands for false negatives, i.e. the number of features that were overseen by the predictor and FP stands for false positives, i.e. the number of features that were predicted but not in agreement with the reference annotation.
Sensitivity and specificity were measured for the features gene (i.e. only a gene structure that was predicted correctly including the exact positions of all CDS exons was counted as TP), exon (i.e. only exons that were predicted correctly were counted as TP) and nucleotide (i.e. every correctly predicted nucleotide was counted as TP).

Hawaiian CB4856 males were crossed into EG1000 dpy-5(e61) I; rol-6(e187) II; lon-1(e1820) III. Fifty Dpy animals and fifty non-Dpy animals from among the self-progeny of EG1000/CB4856 heterozygote hermaphrodites were picked into separate tubes, each containing 20 μL single-worm lysis buffer (50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl₂, 0.45% IGEPAL CA-630, 0.45% Tween 20, 0.01% (w/v) gelatin, 60 ug/ml proteinase K). A further 96 Dpy animals were picked to individual plates for use in interval mapping (see below). They were lysed by freezing at -80°C followed by incubation at 65°C 1 hour and proteinase was inactivated by incubation at 95°C 15 minutes. The Dpy lysate DNA templates were then added to a PCR master mix containing 424 μL water, 52 μL 10X PCR buffer (10X: 22.5 mM MgCl₂, 500 mM Tris-HCl, 140 mM (NH₄)₂SO₄, pH 9.2 at 25°C), 10.4 μL 10 mM dNTPs, and 3.12 μL Taq (5 units/μl). A similar mix was made with the 50 non-Mutant animals. 9.8 μL of the mutant mix or the non-mutant mix was aliquoted into alternate rows of a 96-well PCR plate (Figure 1A). Primer pairs were arrayed into a microtiter plate at 10 μM each primer, so that neighboring rows contain duplicate pairs, and pin-replicated into the master mix. PCR reactions were done using the cycling conditions: 2' 94°C, 35 cycles of (15" 94°C, 45" 60°C, 1' 72°C), 5' 72°C. After amplification, PCR products were digested in the plate with the restriction enzyme DraI in a final volume of 16 μL (10 μL PCR product, 4.15 μL H2O 1.6 μL 10X DraI buffer (New England Biolabs), 0.25 μL DraI (10 units/μL, New England Biolabs)). This was accomplished by adding 6 μL of the enzyme plus enzyme buffer mix to each well using a multi-channel pipette followed by brief centrifugation in a Sorval RT6000D centrifuge with an H1000B rotor. Digestion reactions were incubated at 37°C at least 4 hours. Samples were then loaded onto a 2.5% agarose gel using an 8-channel pipette. The resulting gel displays all 48 SNP markers, from left to right and from chromosome I to X. Each Mutant SNP is next to its non-Mutant control, so that the whole genome can be quickly scanned for linkage.

To verify the expression data of SmPRMTs in the different cell types of adult S. mansoni worms, single-cell RNAseq (scRNAseq) data were obtained from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/, BioProject PRJNA611783, SRA SRP252217; https://www.collinslab.org/schistocyte/; Wendt et al., 2020 (link)). We also verified Smcarm1 expression in clusters of cells identified in schistosomula at the cellxgene platform¹ (Diaz Soria et al., 2020 (link)). The RDS file containing the expression data in the different cell types was loaded in the R software (v4.1.2; R Core Team, 2020 ) using the Seurat package (v4.1.1; Satija et al., 2015 (link)) and used to build a heatmap with the package ComplexHeatmap (v2.10.0; Gu et al., 2016 (link)). Additionally, we checked the SmPRMTs expression in a publicly available RNAseq dataset (Protasio et al., 2017 (link)) retrieved from WormBase ParaSite as counts of aligned reads per run per gene. Differential expression analysis was carried out using DESeq2 (v. 2_1.38.2; padj < 0.05; Love et al., 2014 (link)) and we used the pheatmap package (v1.0.12; Kolde, 2015 ) in R (v4.1.2; R Core Team, 2020 ) to construct a heatmap representing the log2 fold change for 18-, 28-, 35-, and 38-day post-infection (dpi) relative to 21 dpi.

Seven days after dsRNA exposure, 1,000 schistosomula were separated for RNA extraction and relative expression analysis by quantitative real-time PCR (RT-qPCR). Schistosoma mansoni cytochrome C oxidase I gene (SmcoxI—Smp_900000) was used as the internal control gene. RNA extractions were performed using the TRIzol Reagent method followed by purification with the RNeasy Mini Kit (Qiagen), according to the manufacturer’s guidelines. RNA samples were treated with the TURBO DNA-free kit (Ambion) to remove residual genomic DNA, quantified using the Nanodrop Spectrometer ND-1000, and stored at −70°C.
For adults, for 7 days, two worm pairs per day were removed and macerated with TRIzol Reagent for RNA extraction as described previously. Experiments were performed in four biological replicates.
The cDNAs were synthesized with equal amounts of the extracted RNAs using the SuperScript II Reverse Transcriptase (Invitrogen), with oligo(dT)18 following the manufacturer’s protocol. Primers for qPCR analysis were designed using the Primer 3 program.³ Primer efficiencies were estimated by titration analysis to be 100 ± 5% (data not shown), and the specificity was verified by the melting curve. qPCR reactions were performed on 7500 Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM of each primer in a final volume of 25 μl. Internal controls to evaluate genomic DNA contaminations (RNA samples) and reagent purity (no cDNA) were included in all analyses. The 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used for relative quantification and normalized with SmcoxI. Transcript levels were expressed as a percentage of difference relative to the unspecific (GFP) or negative control.