1. S1 Supporting information Bioinformatic workflow and quality of the metrics
Number of slides: 10

2. RNA-seq: BIOINFORMATIC PIPELINE
Cluster 3.0 / JavaTreeView (v1.1.6r4)
Construction of functional clusters
Pick Random (v1.0.0*)
Standardization of read number to analyse
Cufflinks (v2.1.1)
Exon-intron structures
Cuffmerge (v.1.0.0)
Merger of the 21 exon-intron structures
Htseq (v0.6.1p1)
Quantification of read abundance
RNA-seq: BIOINFORMATIC PIPELINE
cDNA Library construction
PolyA
Single strand
50 nucleotides
Sequencing
HiSeq2000 (Illumina)
SBS technique
FastQ groomer (v1.0.4*)
Fastqsanger format check
FASTX-Toolkit (v1.0.0*)
(1) Quality statistics
(2) Quality score boxplot
(3) Nucleotide distribution chart
Tophat (v2.0.9) with the aligner Bowtie (v )
Mapping on S. mansoni genome version 5.2
BAM
File
SAM
BAM-to-SAM (v0.1.18)
Filter SAM (v1.0.0*)
Deletion of unmapped reads
New S.mansoni transcriptome
Used as reference
Sex-biased genes per developmental stages
QUALITY CHECK
MAPPED READS SAMPLING
DE NOVO TRANSCRIPTOMEASSEMBLY
IDENTIFICATION OF SEX-BIASED GENES
FUNCTIONAL ANALYSES
Blast2GO (v2.6.4)
Male and female specific biological pathways through S. mansoni lifecycle
Blastx (v2.2.30)/ AmiGO (v1.8) / GeneDB
De novo annotation of the 100 best sex-biased genes per stage
STRUCTURAL ANALYSES
IGV (v2.3.16)
Blat (v34)
Cuffcompare (v2.2.1)
Exon/Intron structure genome v5.2 vs de novo transcriptome
DEseq (v1.12.1 )
Assessment of differences in gene expression
* Galaxy tool version
S3 – 2/10
Cluster
Analysis candidate genes expression variation through S. mansoni lifecycle

3. RNA-seq: QUALITY OF THE METRICS

4. RNA-seq: REPLICATE CLUSTERING
♀ 1
♀ 2
♂ 1
♂ 2
♀ 1
♀ 2
♂ 1
♂ 2
♀ 1
♀ 2
♂ 1
♂ 2
♀ 1
♀ 1
♀ 1
♀ 2
♀ 2
♀ 2
♂ 1
♂ 1
♂ 1
♂ 2
♂ 2
♂ 2
Cercariae
Schistosomula s#2
Adult worms
Schistosomula s#1
♂ 1
♀ 1
♂ 2
♀ 2
Schistosomula s#3
♂ 1
♀ 1
♂ 2
♀ 2
100%
0% identity
♀1 & ♀2 female duplicates
♂1 & ♂2 male duplicates
DESeq package (v1.12.1)
S3 – 4/10

5. RNA-seq: HEATMAPS (100 best P-values per stage)
♂ 1
♀ 1
♂ 2
♀ 2
Cercariae
♂ 1
♂ 2
♀ 1
♀ 2
Schistosomula s#1
♂ 1
♀ 1
♂ 2
♀ 2
Schistosomula s#2
♂ 1
♀ 1
♂ 2
♀ 2
Schistosomula s#3
♂ 1
♀ 1
♂ 2
♀ 2
Adult worms
DESeq package (v1.12.1)
S3 – 5/10

6. Proportion of categories
Quality analysis of the de novo transcriptome
Description of the type of matches between the Cufflinks transcripts (XLOC) and the reference transcripts (Smp_ID v5.2)
Number of XLOC
Proportion of XLOC
Match categories
Proportion of categories
Complete match of intron chain
6642
19,11%
Smp overlap
27,86%
Contained
113
0,33%
Potentially novel isoform (fragment): at least one splice junction is shared with a reference transcript
2417
6,95%
Single exon transfrag overlapping a reference exon and at least 10 bp of a reference intron, indicating a possible pre-mRNA fragment.
272
0,78%
Generic exonic overlap with a reference transcript
239
0,69%
A transfrag falling entirely within a reference intron
6613
19,03%
Intronic transcript
Possible polymerase run-on fragment (within 2Kbases of a reference transcript)
1466
4,22%
Others
7,72%
Exonic overlap with reference on the opposite strand
1193
3,43%
An intron of the transfrag overlaps a reference intron on the opposite strand (likely due to read mapping errors) 24
0,07%
Unknown, intergenic transcript
15776
45,39%
Intergenic
Repeat. Currently determined by looking at the soft-masked reference sequence and applied to transcripts where at least 50% of the bases are lower case
0,00% -
(.tracking file only, indicates multiple classifications)
Cuffcompare
(Cufflinks v2.2.1)
*Source of the S. mansoni genome reference: ftp://ftp.sanger.ac.uk/pub/pathogens/Schistosoma/mansoni/Latest_assembly_annotation_others/add_utrs.gff
S3 – 6/10

7. ChIP-seq: QUALITY OF THE METRICS
Male adults
Female adults
Male cercariae
Female cercariae
Unbound_1
Unbound_2
H3K27Me3_1
H3K27Me3_2
Raw data
Groomed
QC passed
Aligned (=1)
Aligned (=1) %
53,40%
50,69%
56,29%
53,17%
47,07%
49,55%
48,14%
50,83%
54,15%
49,83%
59,74%
56,70%
43,18%
48,00%
49,05%
52,95%
Aligned (>1)
Aligned (>1) %
42,33%
45,89%
39,21%
42,74%
46,01%
45,67%
43,25%
42,45%
46,46%
36,18%
39,57%
52,27%
47,85%
46,43%
42,11%
Total mapping %
95,72%
96,59%
95,51%
95,91%
95,07%
95,56%
93,81%
94,07%
96,60%
96,29%
95,92%
96,27%
95,45%
95,85%
95,48%
95,06%
Used for peak calling
Number of peaks
N.A.
8363
6947
14 302
5 697
7 116
11 382
5 044
4 719
S3 – 7/10

8. ChIP-seq: REPLICATE CONSISTENCY OF EPICHIP ANALYSIS
TSS
H3K27Me3 enrichment
Male cercariae, replicate 1
Male cercariae, replicate 2
Female cercariae, replicate 1
Female cercariae, replicate 2
Female cercariae, replicate 3
Position on the gene (bases)
TSS
H3K27Me3 enrichment
Position on the gene (bases)
Male adults, replicate 1
Male adults, replicate 2
Female adults, replicate 1
Female adults, replicate 2
Female adults, replicate 3
S3 – 8/10
TSS = Transcription Start Site

9. ChIP-seq: Male and Female H3K27Me3 enrichments, depending on the developmental stages.
A: Males
Cercariae
Adults
-1000
0 = TSS
+5000
0.5
0.6
0.7
0.8
0.9 1
B: Females
Cercariae
Adults
-1000
0 = TSS
+5000
0.5
0.6
0.7
0.8
0.9 1
Position on the gene (bases)
S3 – 9/10
TSS = Transcription Start Site

10. Statistical test of EpiChIP profile differences
Comparison between sexes
Extreme Differences
Z value p
Cercariae
76,50%
41,91
<0,001
Adults
10,70%
5,862
Comparison between stages
Males
56,30%
30,811
Femelles
25,60%
13,997
All pairs of comparison were significant (Kolmogorov-Smirnov two sample tests; p<0.001). The extreme differences given by the Kolmogorov-Smirnov two sample tests show that: (i) The difference between adult male and adult female distributions is low (10.7% of maximum difference) compare to cercarial stage (76.5% of maximum difference). (ii) The difference in chromatin structural changes from cercariae to adult is twice in males compare to females (25.6% of maximum difference for females vs 56.3% of maximum difference for males).
S3 – 10/10