Gossypium raimondii (D5) 'D5-8' genome ISU_v1
Materials and Methods
DNA was extracted from leaves of D5-8. Samples obtained from Iowa State University, Ames, IA were prepared at the USDA-ARS GBRU core facility using Accel-NGS 2S PCR-Free (Product number 20024 with adapter set 26396, Swift Biosciences, Ann Arbor,MI, USA). Library sizes were validated on the Agilent TapeStation 2200 High Sensitivity D1000 Assay (Part No. 5067-5584, Agilent Technologies, Santa Clara, CA, USA) and assayed for concentration prior to equimolar pooling by a KAPA Library Quantification Kit (Product number KK4854, Kapa Biosystems, Inc., Wilmington, MA, USA) on a qPCR instrument (LightCycler 96, Roche Applied Science, Indianapolis, IN, USA). Each pool was clustered onboard an Illumina HiSeq2500 DNA sequencer with a HiSeq PE (pairedend) Rapid v2 flowcell clustering kit (Product number PE-402- 4002, Illumina, San Diego, CA, USA) and sequenced as 2 x 100 bp with the HiSeq Rapid SBS Kit v2 (Product number FC- 402-4021, Illumina, San Diego, CA, USA). Reads were trimmed and filtered with Trimmomatic v0.32 (Bolger et al. 2014) with the following options: 1) sequence adapter removal, 2) removal of leading and/or trailing bases when the quality score (Q) <28, 3) removal of bases after average Q < 28 (8 nt window) or single base quality <10, and 4) removal of reads <85 nt. Detailed parameters can be found at https://github.com/IGBB/D_Cottons_USDA, last accessed December 18, 2018.
About the assembly
Trimmed data were independently assembled for each species via ABySS v2.0.1 (Simpson et al. 2009), using every 5th kmer value from 40 through 100. A single assembly with the highest E-size (Salzberg et al. 2012) was selected for each species and subsequently annotated with MAKER v2.31.6 (Holt and Yandell 2011) using evidence from: 1) the NCBI G. raimondii EST database (Udall et al. 2006), 2) G. raimondii reference genome predicted proteins, as hosted by CottonGen.org (Paterson et al. 2012), and 3) three ab initio gene prediction programs, that is Genemark v4.30 (Borodovsky et al. 2003), SNAP v2013-11-29 (Korf 2004), and Augustus v3.0.3 (Stanke et al. 2006). Both the SNAP and Augustus models were trained using BUSCO v2.0 (Simão et al. 2015). Chromosomer version 0.1.3 (Tamazian et al. 2016), a reference-assisted scaffolder, was used to scaffold the selected assemblies against the gold standard G. raimondii genome. MAKER v2.31.6 (Holt and Yandell 2011) was used to transfer the previous annotations to the Chromosomer-based scaffolds by rerunning MAKER and using the transcripts from the original annotation as evidence. Assemblies are also available under NCBI PRJNA488266.
Grover, et al. Insights into the evolution of the New World diploid cottons (Gossypium, subgenus Houzingenia) based on genome sequencing. Genome Biol Evol. 2019 Jan 1;11(1):53-71.
Additional information about this analysis:
Functional annotation files for the Gossypium raimondii ISU Genome v1.0 are available for download below. The Gossypium raimondii ISU Genome v1.0 proteins were analyzed using InterProScan in order to assign InterPro domains and Gene Ontology (GO) terms. Pathways analysis was performed using the KEGG Automatic Annotation Server (KAAS).
The Gossypium raimondii D5 ISU v1.0 genome gene prediction files are available in FASTA and GFF3 formats.
Homology of the Gossypium raimondii ISU Genome v1.0 proteins was determined by pairwise sequence comparison using the blastp algorithm against various protein databases. An expectation value cutoff less than 1e-9 was used for the NCBI nr (Release 2018-05) and 1e-6 for the Arabidoposis proteins (Araport11), UniProtKB/SwissProt (Release 2019-01), and UniProtKB/TrEMBL (Release 2019-01) databases. The best hit reports are available for download in Excel format.
Marker alignments were performed by the CottonGen Team of Main Bioinformatics Lab at WSU. The alignment tool 'BLAT' was used to map marker sequences from CottonGen to the Gossypium raimondii ISU me assembly. Markers required 90% identity over 97% of their length. For SSRs & RFLPs, gap size was restricted to 1000bp or less with less than 2 gaps. For dbSNPs and Indels gap size was restricted to 2bp with less than 2 gaps. The available files are in GFF3 format. Markers available in CottonGen are linked to JBrowse.
Transcript alignments were performed by the CottonGen Team of Main Bioinformatics Lab at WSU. The alignment tool 'BLAT' was used to map transcripts to the G. raimondii genome assembly. Alignments with an alignment length of 97% and 90% identify were preserved. The available files are in GFF3 format.