DOI: 10.5061/dryad.f4qrfj6xh
title: Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence

description: Abstract Shrimp are a valuable aquaculture species globally; however,
 disease remains a major hindrance to shrimp aquaculture sustainability and
 growth. Mechanisms mediated by endogenous viral elements have been
 proposed as a means by which shrimp that encounter a new virus start to
 accommodate rather than succumb to infection over time. However, evidence
 on the nature of such endogenous viral elements and how they mediate viral
 accommodation is limited. More extensive genomic data on Penaeid shrimp
 from different geographical locations should assist in exposing the
 diversity of endogenous viral elements. In this context, reported here is
 a PacBio Sequel-based draft genome assembly of an Australian black tiger
 shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft
 genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9%
 of the projected genome size. The genome repeat content (61.8% with 30%
 representing simple sequence repeats) is almost the highest identified for
 any species. The functional annotation identified 35,517 gene models, of
 which 25,809 were protein-coding and 17,158 were annotated using
 interproscan. Scaffold scanning for specific endogenous viral elements
 identified an element comprised of a 9,045-bp stretch of repeated,
 inverted, and jumbled genome fragments of infectious hypodermal and
 hematopoietic necrosis virus bounded by a repeated 591/590 bp host
 sequence. As only near complete linear ∼4 kb infectious hypodermal and
 hematopoietic necrosis virus genomes have been found integrated in the
 genome of P. monodon previously, its discovery has implications regarding
 the validity of PCR tests designed to specifically detect such linear
 endogenous viral element types. The existence of joined inverted
 infectious hypodermal and hematopoietic necrosis virus genome fragments
 also provides a means by which hairpin double-stranded RNA could be
 expressed and processed by the shrimp RNA interference machinery.
description: <strong>Shrimp
 breeding and selection for sequencing:</strong> A second-generation (G2) male
 <em>Penaeus monodon</em> that had undergone a single cycle of
 inbreeding was selected for genomic sequencing. The original wild-caught
 broodstock were collected from a Queensland east coast location
 (approximately 17.3°S, 146.0°E) in September 2013. In October 2013, 14
 first-generation (G1) families were produced from the brood stock at
 Seafarm Flying Fish Point hatchery (approximately 17.5°S, 146.1°E). In
 February 2015, pleopod tissue was sampled from 50 female and 50 male G1
 broodstock. These tissues were genotyped (using 2 x 60 SNP panels (Sellars
 et al. 2014) to identify the parental origin of each broodstock and to
 select related mating pairs to generate the inbred G2 progeny. In August
 2015, groups of 50 juvenile males from 5 inbred G2 families were
 euthanized to collect muscle tissue from the first abdominal segment for
 sequencing and the second most anterior pair of pleopods for genotyping.
 These tissues, as well as the remainder of each shrimp (archived source of
 tissue for sequencing) were snap frozen under dry ice pellets and stored
 at -80°C. Each shrimp was then genotyping using the 120-SNP panel (Sellars
 et al. 2014) and a genome-wide SNP assay based on DArTSeq (Guppy et al.
 2020). After ranking the 50 males based on inbreeding coefficient (F) and
 multi-locus heterozygosity (MLH) data from the 120-SNP panel, the
 individual (named Nigel) with the highest inbreeding coefficient was
 chosen for genomic sequencing. The choice was confirmed using a
 genome-wide SNP assay based on DArTSeq of the top five inbred shrimp based
 on the 120-SNP panel which recovered the same ranking (Nigel: MLH of 0.231
 and F of 0.271). <strong>DNA extraction,
 library preparation and genome sequencing:</strong> Multiple extraction methods were
 trialed to generate intact high-quality genomic DNA from stored muscle
 tissue of the single selected inbred shrimp. All DNA extractions and
 sequencing runs were carried out at the Australian Genome Research
 Facility (AGRF), Melbourne, Australia. For Illumina sequencing, the
 MagAttract HMW DNA kit (QIAGEN) was used and PCR-free fragment shotgun
 libraries were prepared using the ‘with-bead pond library’ construction
 protocol described by Fisher et al. (Fisher et al. 2011) with some
 modifications. The library was sequenced on two HiSeq 2500 lanes using a
 250 bp PE Rapid sequencing kit (Illumina). The same DNA was also used to
 create a 10X Genomics Chromium library as per manufacturer instructions,
 which was sequenced on two HiSeq 2500 lanes using a 250 bp PE Rapid
 sequencing kit. For PacBio sequencing, the following DNA extraction
 methods were used with varying success: MagAttract HMW DNA kit (QIAGEN),
 Nanobind HMW Tissue DNA kit-alpha (Circulomics), and
 CTAB/Phenol/Chloroform. Libraries were prepared using the SMRTbell
 Template Prep Kit 1.0 (PacBio), loaded using either magbeads or diffusion,
 and sequenced using the Sequel Sequencing Kits versions 2.1 and 3.0 on a
 PacBio Sequel (Supplementary Table 1). The same muscle tissue was also
 used to prepare three Dovetail Hi-C libraries according to
 manufacturer's instructions. Two libraries were sequenced on a shared
 lane of a NovaSeq S1 flow cell, and a third library was sequenced on one
 lane of a NovaSeq SP flow cell, with both sequencing runs generating 100
 bp paired-end reads. <strong>Genome
 assembly:</strong> The quality of the initial
 short-read genome assemblies using either DISCOVAR <em>de
 novo</em> (Weisenfeld et al. 2014) with Illumina data, or Supernova
 (Weisenfeld et al. 2017) with 10X Genomics Chromium data was poor. The
 most contiguous assembly was achieved using wtdbg2/redbean (Version 2.4,
 Ruan and Li 2019) with 75 X times coverage of PacBio data, setting the
 estimated genome size to 2.2 Gb, but without using the wtdbg2 inbuilt
 polishing. The raw assembly was subjected to two rounds of polishing using
 the PacBio subreads data in arrow (Version 2.3.3, github.com/PacificBiosciences/GenomicConsensus) and one round of polishing using the Illumina short-read data in pilon (Version 1.23, Walker et al. 2014). Scaffolds were constructed in two steps. Medium-range scaffolding carried out using 10X Genomics Chromium data with longranger (Version 2.2.2, https://support.10xgenomics.com/genome-exome/software/downloads/latest) and ARCS (Version 1.0.6, Yeo et al. 2017), while long-range scaffolding was performed using dovetail Hi-C data, and intra- and inter-chromosomal contact maps were built using HiC-Pro (Version 2.11.1, Servant et al. 2015) and SALSA (commit version 974589f, Ghurye et al. 2017). This genome assembly was then submitted to NCBI GenBank, which required the removal of two small scaffolds and the splitting of one scaffold. The overall quality of the final V1.0 genome was assessed using BUSCO, and through mapping of RNA-seq, and Illumina short-reads using HiSAT2 (version 2.1.0, Kim et al. 2019). <strong>Repeat annotation:</strong> Repeat content was assessed with <em>de novo</em> searches using RepeatModeler (V2.0.1) and RepeatMasker (V4.1.0) via Dfam TE-Tools (V1.1, https://github.com/Dfam-consortium/TETools) within Singularity (V2.5.2, Kurtzer et al. 2017). Additionally, tandem repeat content was determined using Tandem Repeat Finder (V4.0.9, Benson 1999) within RepeatModeler. Analyses and plotting of interspersed repeats were carried out as per Cooke<em> et al.</em> (2020, github.com/iracooke/atenuis_wgs_pub/blob/master/09_repeats.md). Additionally, the genomes of the Black tiger shrimp (Thai origin, www.biotec.or.th/pmonodon; Kim et al. 2019), Whiteleg shrimp (<em>P. vannamei</em>, NCBI accession: QCYY00000000.1; Zhang et al. 2019), Japanese blue crab (<em>Portunus trituberculatus</em>, gigadb.org/dataset/100678; Tang et al. 2020), and Chinese mitten crab (<em>Eriocheir japonica sinensis</em>, NCBI accession: LQIF00000000.1) were run through the same analyses for comparison. <strong>Gene prediction and annotation:</strong> In order to generate an RNA-seq based transcriptome, raw data from a previous study (NCBI project PRJNA421400; Huerlimann et al. 2018) was mapped to the masked genome using STAR (Version 2.7.2b; Dobin et al. 2013), followed by Stringtie (Version 2.0.6; Pertea et al. 2015) (Supplementary Table 2). Additionally, the IsoSeq2 pipeline (PacBio) was used to process the ISO-seq data generated in this study (Supplementary Table 2). Finally, the genome annotation was carried out in MAKER2 (v2.31.10; Campbell et al. 2014; Cantarel et al. 2008; Holt and Yandell 2011) using the assembled RNA-seq and ISO-seq transcriptomes together with protein sequences of other arthropod species.
description: This file contains the genome annotation of the Australian
 Penaeus monodon genome deposited on NCBI. Raw and
 assembled sequence data generated by this study have been deposited in
 GenBank BioProject PRJNA590309, BioSample SAMN13324362. PacBio and
 Illumina raw data can be found under accession numbers
 SRR10713990-SRR10714025. The final scaffolded assembly can be found under
 accession JAAFYK000000000. RNA-seq data used for annotation originated
 from an earlier study (Huerlimann et al. 2018).