Computational protocol: Sequencing and de novo transcriptome assembly of Anthopleura dowii Verrill (1869), from Mexico

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Protocol publication

[…] RNA was isolated using the SV Total RNA Isolation System (Promega) following the protocol provided by the manufacturer. Briefly, 30 mg of tentacle tissue were manually macerated to homogeneity with a Kontes microtube pellet pestle rod (Daigger) in a 1.5 ml microcentrifuge tube with 175 μl of the provided RNA Lysis Buffer. After dilution with 350 μl of the RNA Dilution Buffer the sample was heated for 3 min at 70 °C. Cellular debris were then discarded by centrifugation. The cleared lysate was mixed with 95% ethanol and transferred to one of the spin baskets supplied with the kit. After washing with the RNA Wash Solution, the sample was treated with the provided DNAse for 15 min and then washed twice with the RNA Wash Solution. After centrifugation, total RNA was recovered in nuclease-free water. The RNA was quantitated with a Nanodrop 1000 (Thermo Scientific) and its integrity was confirmed using a 2100 Bioanalyzer (Agilent Technologies). RIN values (RNA integrity number) of 8.5 were obtained, indicating that the RNA had the needed quality to proceed to library construction and high-throughput sequencing.A complementary DNA (cDNA) library was constructed from the obtained total RNA, using the Illumina TruSeq Stranded mRNA Sample Preparation Kit, following the protocol provided by the supplier. Automated DNA sequencing was performed at the Massive DNA Sequencing facility in the Institute of Biotechnology (Cuernavaca, Mexico) with a Genome Analyzer IIx (Illumina), using a 72 bp paired-end sequencing scheme over cDNA fragments ranging in size of 200–400 bp. Each library consisted of two fastq files (forward and reverse reads), from which the adaptors were clipped-off. The quality of cleaned raw reads was verified with the FastQC program (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). [...] A. dowii Verrill (1869) RNA-seq raw data was de novo assembled using Trinity , a program based in De Brujin graphs for the assembly of short reads. Trinity was executed using default parameters for the assembly of paired-end reads. For mapping and abundance estimation, reads were mapped with Bowtie , using the reconstructed transcriptome as a reference. Abundance of transcripts were estimated by RSEM , as described in the Trinity protocol . Global GC content of the sequences was determined using Emboss GeeCee tool .A total of 70,097,332 raw reads from Illumina technology were produced for A. dowii Verrill (1869) transcriptome sequencing. These reads were assembled by Trinity pipeline resulting in 72,684 contigs with a N50 = 1179 bp, average length of 707 bp. (). This work provides the first transcriptome assembly for the sea anemone A. dowii Verrill (1869). The information presented here may be useful to identify new molecules for biotechnology and pharmaceutical relevance. […]

Pipeline specifications

Software tools FastQC, Trinity, Bowtie, RSEM, EMBOSS
Application RNA-seq analysis