diff --git a/README.md b/README.md index 7c903c4..6deb33d 100644 --- a/README.md +++ b/README.md @@ -352,6 +352,179 @@ Cantalapiedra et al. 2021) with the following options: The other options were set by default. +## Additional steps + +**Over-represented sequences analysis**: We extracted the +overrepresented sequences identified using FastQC and aligned them to +the Nucleotide database. The result is showed in the Table S2. + +``` r +# Attach the packages +library(fastqcr) +library(dplyr) +library(Biostrings) + +# Extract the overrepresented sequences and their abundance +overseq_tbl <- full_join( + qc_plot(qc_read("sample_1_fastqc.zip"), "Overrepresented sequences") %>% select(Sequence, Count) %>% rename("frcts" = Count), + qc_plot(qc_read("sample_2_fastqc.zip"), "Overrepresented sequences") %>% select(Sequence, Count) %>% rename("rvcts" = Count) + ) + +# Export overrepresented sequences +overseq <- DNAStringSet(overseq_tbl %>% pull(Sequence)) +names(overseq) <- paste0("ELVIRA_OVERSEQ-", 1:nrow(overseq_tbl)) +writeXStringSet(x = overseq, filepath = "sample.overseq.fna", append = FALSE) +``` + +``` bash +# Align the overrepresented sequences to the Nucleotide database +blastn \ + -num_threads $SLURM_NTASKS \ + -task blastn-short \ + -db nt \ + -query sample.overseq.fna + -out sample.overseq.nt.tsv \ + -evalue 1e-3 \ + -outfmt 6 \ + -perc_identity 75 \ + -max_target_seqs 1 \ + -max_hsps 1 +``` + +**Transcriptome fragmentation**: We checked if the transcript were +assembled completely and the proteins coverage (we required the +alignment obtained in the STEP 7). + +``` r +# Calculate fragmentation and protein coverage +trans_frag <- check_completeness( + prot_seqs = "sample.good.rmdup.slugs.faa", + prot_blast = "sample.good.rmdup.slugs.nr.tsv" +) + +# Export the result +write_delim(trans_frag, file = "sample.fragmentation.tsv", delim = "\t", append = FALSE, col_names = TRUE) +``` + +**Orthogroups detection**: We detected the different orthogroups using +OrthoFinder (Emms and Kelly 2019). After, these sequences were aligned +using MAFFT (Katoh et al. 2002). OrthoFinder is not reporting the final +species tree correctly (Issue +\#732)\[\] so, we +had to build it using an alternaltive approach. + +``` bash +# Create a dump directory to store the different transcriptomes +mkdir ref_sequences + +# Run OrthoFinder +orthofinder \ + -f ref_sequences \ + -a $SLURM_NTASKS \ + -M msa \ + -A mafft \ + -T raxml-ng \ + -o slugs_ortphy +``` + +**Building the species tree**: + +``` bash +# Estimate the substitution model +modeltest-ng \ + --force \ + --processes $SLURM_NTASKS \ + --datatype aa \ + --input slugs_ortphy/*/MultipleSequenceAlignments/SpeciesTreeAlignment.fa \ + --output SpeciesTreeAlignment.model \ + --topology ml \ + --frequencies e \ + --model-het f \ + --template raxml + +# Build the ML tree +raxml-ng \ + --redo \ + --threads $SLURM_NTASKS \ + --all \ + --check \ + --msa slugs_ortphy/*/MultipleSequenceAlignments/SpeciesTreeAlignment.fa \ + --model MODEL \ + --blopt nr_safe \ + --bs-trees 10000 +``` + +**Orthogroups gene-ontology enrichment** + +``` r +# Attach the libraries +library(topGO) +library(readr) +library(dplyr) +library(stringr) + +# Load the Orthogroups information +cnames <- c("Orthogroup", "ACA", "ECH", "ECO", "ECR", "EOR", "ETI", "EVI", "OVI", "POC") +orthogroups <- read_delim(file = "orthofinder_dir/Orthogroups.tsv", delim = "\t", col_names = cnames, skip = 1) + +# Load ELVIRA annotation +txannotation <- readMappings(file = "sample.good.rmdup.slugs.trans2go.tsv", sep = "\t", IDsep = ";") +``` + +``` r +# Pick up the genes of interest +# KLEPTOPLASTY +txsubset <- orthogroups %>% + filter(is.na(ACA), is.na(OVI), !is.na(ECO), !is.na(EOR), !is.na(ECR), !is.na(ETI), !is.na(ECH), !is.na(EVI), !is.na(POC)) %>% + pull(EVI) + +# LONG-TERM RETAINERS +txsubset <- orthogroups %>% + filter(is.na(ACA), is.na(OVI), is.na(ECO), is.na(EOR), !is.na(ECR), !is.na(ETI), !is.na(ECH), !is.na(EVI), !is.na(POC)) %>% + pull(EVI) + +# LTR ULVOPHYCEAE-FEEDER +txsubset <- orthogroups %>% + filter(is.na(ACA), is.na(OVI), is.na(ECO), is.na(EOR), !is.na(ECR), !is.na(ETI), is.na(ECH), !is.na(EVI), !is.na(POC)) %>% + pull(EVI) + +# Select the genes of interest +txsubset <- unlist(str_split(string = txsubset, pattern = ", ")) +txsubset <- factor(as.integer(names(txannotation) %in% txsubset)) +names(txsubset) <- names(txannotation) +``` + +``` r +# Perform GO-term enrichment +# Biological Process +bp_godata <- new("topGOdata", ontology = "BP", allGenes = txsubset, annot = annFUN.gene2GO, gene2GO = txannotation) +bp_enrichment <- runTest(object = bp_godata, algorithm = "weight01", statistic = "t") +bp_enrichment <- GenTable(object = bp_godata, pvalue = bp_enrichment, topNodes = 2583) + +# Cellular Component +cc_godata <- new("topGOdata", ontology = "CC", allGenes = txsubset, annot = annFUN.gene2GO, gene2GO = txannotation) +cc_enrichment <- runTest(object = cc_godata, algorithm = "weight01", statistic = "t") +cc_enrichment <- GenTable(object = cc_godata, pvalue = cc_enrichment, topNodes = 683) + +# Molecular Function +mf_godata <- new("topGOdata", ontology = "MF", allGenes = txsubset, annot = annFUN.gene2GO, gene2GO = txannotation) +mf_enrichment <- runTest(object = mf_godata, algorithm = "weight01", statistic = "t") +mf_enrichment <- GenTable(object = mf_godata, pvalue = mf_enrichment, topNodes = 1606) + +# Binding enrichment result +go_enrichment <- tibble(bind_rows( + bp_enrichment %>% mutate("go_class" = "BP"), + cc_enrichment %>% mutate("go_class" = "CC"), + mf_enrichment %>% mutate("go_class" = "MF") +)) %>% + mutate(pvalue = str_replace(string = pvalue, pattern = "< ", replacement = ""), + pvalue = as.numeric(pvalue), + go_class = factor(go_class, levels = c("BP", "CC", "MF"))) + +# Export the result +write_delim(x = go_enrichment, file = "go_enrichment.tsv", delim = "\t", append = FALSE, col_names = TRUE) +``` + # References
@@ -412,6 +585,14 @@ Computational Biology* 7 (10): e1002195.
+
+ +Emms, David M, and Steven Kelly. 2019. “OrthoFinder: Phylogenetic +Orthology Inference for Comparative Genomics.” *Genome Biology* 20 (1): +1–14. + +
+
Haas, Brian J, Alexie Papanicolaou, Moran Yassour, Manfred Grabherr, @@ -448,6 +629,14 @@ Opisthobranchia).” *Bonner Zoologische Beiträge* 55 (3/4): 255–81.
+
+ +Katoh, Kazutaka, Kazuharu Misawa, Kei-ichi Kuma, and Takashi Miyata. +2002. “MAFFT: A Novel Method for Rapid Multiple Sequence Alignment Based +on Fast Fourier Transform.” *Nucleic Acids Research* 30 (14): 3059–66. + +
+
Manni, Mosè, Matthew R Berkeley, Mathieu Seppey, Felipe A Simão, and