DOI: 10.5061/dryad.mcvdncjz5
title: De novo genome assembly of Tectona grandis (Teak) with 2993 scaffolds

description: Teak (Tectona grandis L. f.) is one of the precious bench mark
 tropical hardwood having qualities of durability, strength and visual
 pleasantries. Natural teak populations harbour a variety of
 characteristics that determine their economic, ecological and
 environmental importance. Sequencing of whole nuclear genome of teak
 provides a platform for functional analyses and development of genomic
 tools in applied tree improvement. A draft genome of 317 Mb was assembled
 at 151× coverage and annotated 36, 172 protein-coding genes. Approximately
 about 11.18% of the genome was repetitive. Microsatellites or simple
 sequence repeats (SSRs) are undoubtedly the most informative markers in
 genotyping, genetics and applied breeding applications. We generated
 182,712 SSRs at the whole genome level, of which, 170,574 perfect SSRs
 were found; 16,252 perfect SSRs showed in
 silico polymorphisms across six genotypes suggesting their
 promising use in genetic conservation and tree improvement programmes.
 Genomic SSR markers developed in this study have high potential in
 advancing conservation and management of teak genetic resources.
 Phylogenetic studies confirmed the taxonomic position of the
 genus Tectona within the family Lamiaceae.
 Interestingly, estimation of divergence time inferred that the Miocene
 origin of the Tectona genus to be around 21.4508 million
 years ago.
description: WGS was performed using Illumina HiSeq 2000 platform and Oxford
 Nanopore Technologies MinION device by the Genotypic Technology,
 Bengaluru, India in accordance to standard protocols. Accession 2 was
 selected for the generation of high quality reference genome assembly.
 Accessions 1, 3, 4, 5 and 6 were subjected to low coverage genome
 sequencing to identify polymorphic SSRs. In the case of accession 2, one
 paired end (PE) (150 bp × 2) library of size 300–700 bp, two mate pair
 (MP) libraries (2–4 and 4–6 kb fragments) and one nanopore library with
 genomic DNA (2 μg) were prepared for sequencing. In Illumina HiSeq 2000
 platform, one lane of the flow cell was used for each sequencing library.
 Nanopore sequencing was performed using R9.4 flow cells on a MinION Mk 1B
 device (Oxford Nanopore) with the MinKNOW software (versions 1.0.5–1.5.12)
 and base calling was performed using Albacore 1.1.0 (Oxford Nanopore).
 Template reads were exported as FASTA using poretools version 0.6. In the
 case of other five accessions (1, 3, 4, 5 and 6) one PE library for each
 with the size of 300–700 bp was sequenced at ∼15× coverage through
 Illumina Hiseq 2000 platform. The raw data is uploaded in genome database
 of GenBank (Project id: PRJNA374940).  The Illumina PE raw reads were filtered using FastQC and the raw reads were processed by in-house (Genotypic Technology, Bangaluru, India) ABLT script for low-quality bases and adapters removal. The MP reads were processed using Platanus<sup>24</sup> internal trimmer for adapters and low-quality regions towards 3’-end. The processed PE reads along with MP and nanopore reads were used for contig generation using MaSuRCA v 3.2.2 <em>de novo</em> assembler.<sup>25</sup> To assemble the genome following command was used in MaSuRCA assembler: GRAPH_KMER_SIZE = auto, LIMIT_JUMP_COVERAGE = 300, JF_SIZE = 38000000000, DO_HOMOPOLYMER_TRIM = 1. Scaffolding of the assembled contigs was performed using SSPACE v 2.0.5<sup>26</sup> with processed PE and MP reads followed by gap filling using Gap Closer v 1.12.<sup>27</sup> The genome size was estimated automatically during read computing stage which utilized both the Illuimna and Nanopore reads. Similarly, the low depth Illumina reads generated for five accessions of teak were assembled using accession 2 as reference. The sequenced data was uploaded to the Genome database of GenBank (Project id: PRJNA421422). For a functional overview of draft genome, assembled scaffolds were converted to FASTA formatted sequences, hard masked by RepeatMasker tool (RepeatMasker Open-3.0; www.repeatmasker.org (10 November 2017, date last accessed)). Repeats of <em>Arabidopsis thaliana</em> were used as reference for genome masking. Gene prediction was carried out using Augustus 3.0.2<sup>28</sup> programme and predicted proteins were searched against the Uniprot non-redundant plant protein database (Taxonomy = <em>Viridiplante</em>) with BlastX algorithm with an <em>e</em>-value (<em>e</em>-10) for gene ontology and annotation. Pathway annotation was performed by mapping the sequences obtained from Blast2GO to the contents of the KEGG Automatic Annotation Server (http://www.genome.jp/kegg/kaas/ (10 November 2017, date last accessed).