Computational protocol: Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA seq, identifies germline enriched microRNAs☆

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Protocol publication

[…] Sequencing data are available at NCBI in the Bioproject database with accession number PRJNA322183 (http://www.ncbi.nlm.nih.gov/bioproject/PRJNA322183). Data were generated by Illumina sequencing, with reads cleaned to remove low quality bases and trimmed to remove the adaptors. All reads were aligned against the annotated, non-coding RNA transcripts downloaded from Wormbase ftp://ftp.wormbase.org/pub/wormbase/releases/WS242/species/c_elegans/PRJNA13758/c_elegans. PRJNA13758. WS242.ncrna_transcripts.fa.gz, which contains annotations for 223 miRNAs, 345 snoRNAs, 126 snRNA, 176 lincRNAs, 7980 ncRNAs, 15,365 piRNAs and other RNAs, including tRNAs, and rRNAs. The alignment from the above processed short reads to the non-coding transcripts was conducted using BWA, Burrows-Wheeler alignment (). To obtain the counts for each miRNA, the HTSeq protocol () was applied to the mapping results, which are found in .Statistical tests comparing differential abundance levels between the miRNAs of various populations were carried out using DESeq2, a statistical analysis software available as an R/Bioconductor package (). DESeq2 software was used because it reports consistent results even for experiments with small number of replicates (). For our sequencing results DESeq2 provided much more conservative estimates of statistical significance than did the software package edgeR (). The adjusted p-value (q-value) of <0.05 was used as the cutoff for differential expression with statistical significance. q-values are adjusted p-values, used with the Benjamini-Hochberg procedure (). http://www.stat.purdue.edu/~doerge/BIOINFORM.D/FALL06/Benjamini%20and%20Y%20FDR.pdf. […]

Pipeline specifications

Software tools BWA, HTSeq, DESeq2, edgeR
Databases WormBase
Application Non-coding RNA analysis
Organisms Caenorhabditis elegans