Computational protocol: The house spider genome reveals an ancient whole-genome duplication during arachnid evolution

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Protocol publication

[…] The house spider and bark scorpion are two of 30 arthropod species sequenced as part of the pilot project for the i5K 5000 arthropod genomes project at the Baylor College of Medicine Human Genome Sequencing Center. For all of these species, an enhanced Illumina-ALLPATHS-LG sequencing and assembly strategy enabled multiple species to be approached in parallel at reduced costs. For the house spider, we sequenced five libraries of nominal insert sizes 180 bp, 500 bp, 2 kb, 3 kb, and 8 kb at genome coverages of 39.2x, 35.1x, 19.7x, 49.3x, and 19.3x, respectively (assuming a 1.5 Gb genome size []). These raw sequences have been deposited in the NCBI SRA: BioSample ID SAMN01932302. For the bark scorpion, we sequenced four libraries of nominal insert sizes 180 bp, 500 bp, 3 kb, and 8 kb at genome coverages of 102.1x, 25.6x, 35.2x, and 39.0x, respectively (assuming a 900 Mb genome size). These raw sequences have been deposited in the NCBI SRA: BioSample SAMN02617800.To prepare the 180 bp and 500 bp libraries, we used a gel-cut paired-end library protocol. Briefly, 1 μg of the DNA was sheared using a Covaris S-2 system (Covaris, Inc. Woburn, MA) using the 180 bp or 500 bp program. Sheared DNA fragments were purified with Agencourt AMPure XP beads, end-repaired, dA-tailed, and ligated to Illumina universal adapters. After adapter ligation, DNA fragments were further size-selected on an agarose gel and PCR-amplified for 6 to 8 cycles using the Illumina P1 and Index primer pair and Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The final library was purified using Agencourt AMPure XP beads and quality-assessed by Agilent Bioanalyzer 2100 (DNA 7500 kit) to determine library quantity and fragment size distribution before sequencing.Long mate pair libraries with 2 kb, 3 kb, and 8 kb insert sizes were constructed according to the manufacturer’s protocol (Mate Pair Library v2 Sample Preparation Guide art # 15001464 Rev. A PILOT RELEASE). Briefly, 5 μg (for 2 and 3 kb gap size libraries) or 10 μg (8–10 kb gap size library) of genomic DNA was sheared to the desired size fragments by Hydroshear (Digilab, Marlborough, MA), then end-repaired and biotinylated. Fragment sizes between 1.8 and 2.5 kb (2 kb), 3 and 3.7 kb (3 kb), or 8 and 10 kb (8 kb) were purified from 1% low-melting agarose gel and then circularized by blunt-end ligation. These size-selected circular DNA fragments were then sheared to 400 bp (Covaris S-2), purified using Dynabeads M-280 Streptavidin Magnetic Beads, end-repaired, dA-tailed, and ligated to Illumina PE-sequencing adapters. DNA fragments with adapter molecules on both ends were amplified for 12 to 15 cycles with Illumina P1 and Index primers. Amplified DNA fragments were purified with Agencourt AMPure XP beads. Quantification and size distribution of the final library was determined before sequencing as described above.Sequencing was performed using Illumina HiSeq2000 generating 100 bp paired-end reads. Reads were assembled using ALLPATHS-LG (v35218) [] and further scaffolded and gap-filled using Atlas-Link (v.1.0) and Atlas gap-fill (v.2.2) []. For P. tepidariorum, this yielded an assembly size of 1443.9 Mb with 263,833 contigs with an N50 of 10.1 kb and, after scaffolding and gap closing, 31,445 scaffolds with an N50 of 465.5 kb. Approximately 2416 million reads (96.9x sequence coverage) are represented in this assembly of the P. tepidariorum genome. The assembly has been deposited in the NCBI: BioProject PRJNA167405 (Accession: AOMJ00000000).For the C. sculpturatus this yielded an assembly size of 926.4 Mb with 214,941 contigs with an N50 of 5.1 kb and, after scaffolding and gap closing, 10,457 scaffolds with an N50 of 342.5 kb. The final assembly has been deposited in the NCBI: BioProject PRJNA168116. […]

Pipeline specifications

Software tools ALLPATHS-LG, Atlas-Link
Application De novo sequencing analysis
Organisms Parasteatoda tepidariorum