DOI: 10.5061/dryad.0k6djhb18
title: Genome sequencing of Pachypeltis micranthus Mu et Liu (Hemiptera: Miridae), a potential biological control agent for Mikania micrantha

description: The plant bug, Pachypeltis micranthus Mu et Liu (Hemiptera: Miridae), is a
 potential biological control agent for Mikania micrantha H.B.K.
 (Asteraceae; one of the most invasive weeds worldwide). To date, only a
 few studies have investigated plant bugs. Here, we performed a
 chromosome-level genome assembly of P. micranthus using MGISEQ-2000
 short-read, Nanopore, PacBio long-read, and high-throughput chromosome
 conformation capture (Hi-C) techniques. The assembled genome was 712.72 Mb
 in size, with a contig N50 of 16.84 Mb. Using the Hi-C technique, 71
 scaffolds were assembled into 15 chromosomes, accounting for 99.96%. We
 predicted 11,746 protein-coding genes in P. micranthus with 96.20%
 complete benchmarking universal single-copy orthologs. Phylogenomic
 analysis showed that P. micranthus and two other Miridae bugs (Apolygus
 lucorum and Nesidiocoris tenuis) diverged from the common ancestor
 approximately 200.01 million years ago. Chromosome synteny analysis
 between P. micranthus and A. lucorum indicated high-level synteny. Many
 gene families including chemosensory genes and digestive and
 detoxification enzyme genes—were significantly expanded in the P.
 micranthus genome. These expanded gene families may indicate the bug to
 adapt to the single host plant. This high-quality chromosome-level genome
 assembly provides an invaluable resource for further molecular and
 evolutionary research on mirid bugs and also provides a basis for further
 research on biological control mechanisms for M. micrantha.
description: Male adults of
 <i>Pachypeltis micranthus</i> were collected five days after
 emergence and used for genome sequencing. DNA was extracted from these
 male adult bugs and determined the quality and purity. The qualified DNA
 was used to construct the sequencing library. Next-generation sequencing
 was performed using the MGISEQ-2000 (MGI, China) platform. Oxford Nanopore
 GridION X5 (Oxford Nanopore Technologies, UK) and PacBio Sequel Ⅱ (Pacific
 Biosciences, CA, USA) platforms were used for third-generation sequencing.
 The high-throughput chromosome conformation capture (Hi-C) library was
 sequenced using the Illumina Novaseq (Illumina, FL, USA) platform. Three
 RNA-seq libraries of male adult bugs were constructed to annotate the
 genomic structure of <i>P. micranthus</i>. Sequencing was
 performed using an Illumina HiSeq 2000 (Illumina, FL, USA) platform.
 All sequenced raw data were filtered using Fastp v0.20.0 (Chen, Zhou,
 Chen, & Gu, 2018). We obtained 113.67 Gb of
 MGISEQ-2000 short reads, 75.52 Gb of Nanopore long reads, and 12.36 Gb of
 PacBio long reads, with 154.80X, 98.87X, and 13.75X genome coverage,
 respectively. The filtered Nanopore reads were used for genome assembly
 with the NextDenovo package (https://github.com/Nextomics/NextDenovo). The
 original subreads were self-corrected using the NextCorrect module. In
 total, 13.6 Gb of consistent sequences was obtained and used for
 preliminary genome assembly using the NextGraph module. The preliminary
 assembly was performed with a genome size of 710.81 Mb and a median contig
 length (N50) of 16.82 Mb. Finally, the contigs were corrected three times
 with NextPolish v1.3.0 (Hu, Fan, Sun, & Liu, 2020) using Nanopore
 long reads and refined four times with the Racon module using the
 MGISEQ-2000 short reads. The final polished genome size was 712.72 Mb with
 a contig N50 of 16.84 Mb. Benchmarking Universal Single-Copy Orthologs
 (BUSCO) v4.0.5 (Simao, Waterhouse, Ioannidis, Kriventseva, &
 Zdobnov, 2015), Bwa v0.7.12 (Li & Durbin, 2010) and CEGMA v2
 (Parra, Bradnam, & Korf, 2007) results showed that the genome
 assembly was complete and accurate. Minimap2 v41 (Li, 2016) result
 indicated that the genome wasn’t contaminated. The clean reads of RNA-seq were
 mapped to the <i>P. micranthus</i> genome using Hisat2 v2.1.0
 (Kim, Langmead, & Salzberg, 2015). The fragments per kilobase per
 million (FPKM) values were calculated using StringTie (Pertea et al.,
 2015). Using the three methods of gene prediction (RNA-seq data, de novo
 prediction, and homology detection), we predicted 11,746 protein-coding
 genes with an average transcript length of 32,170.81 bp, an average CDS
 length of 1,515.18 bp, an average exon length of 193.96 bp, an average
 number of 7.82 exons per gene, and an average intron length of 4,496.87
 bp. We annotated
 the tandem repeats (TRs) and transposable elements (TEs) in the
 <i>P. micranthus</i> genome. First, TRs were detected using
 Tandem Repeats Finder (TRF, v4.07) (Benson, 1999) and GMATA v2.2, using
 default parameters (Wang & Wang, 2016). The TRF was used to search
 for TRs, and GMATA was mainly used to search for simple sequence repeats
 (SSRs) of TRs. For TE identification, we constructed a de novo repeat
 library using RepeatModeler v1.0.11, with default parameters (Bedell,
 Korf, & Gish, 2000). Known and novel TEs in the bug genome were
 identified by aligning the proteins against the de novo repeat library and
 the Repbase TE library with RepeatMasker v1.331 (Bedell et al., 2000). In
 total, we generated 375.82 Mb (52.73% of the genome) of repeat sequences
 that were classified into 12 types. Among these, DNA transposons (19.19%
 of the genome) and long interspersed nuclear elements (18.16% of the
 genome) accounted for the majority of the TEs (48.39% of the genome). A
 total of 20,783 SSRs (0.03% of the genome) were identified in the TRs
 (0.02% of the genome). For chromosome-level genome
 assembly, we obtained 112 Gb of raw data. After filtering, 110.96 Gb of
 clean data was generated. The high-quality reads were mapped to the
 assembled genome using Bowtie2 v2.3.2 (sensitive end-to-end) (Langmead
 & Salzberg, 2012). The unique mapped paired-end reads obtained
 were used to identify and retain valid interaction paired reads using
 HiC-Pro v2.8.1 (Servant et al., 2015). Based on the valid interaction
 paired reads, the scaffolds were ordered, oriented, and joined into
 chromosomes using LACHESIS (https://github.com/shendurelab/LACHESIS) (Burton et al., 2013). Finally, we manually adjusted the position and orientation errors showing significant chromatin interactions. a total of 71 scaffolds (total length—712.46 Mb) were anchored, ordered, and oriented into 15 chromosomes (total length—707.51 Mb), accounting for 99.27% of the assembled <i>P. micranthus</i> genome. <b>References</b> Bedell, J. A., Korf, I., & Gish, W. (2000). MaskerAid: a performance enhancement to RepeatMasker. <i>Bioinformatics, 16</i>(11), 1040-1041. doi:10.1093/bioinformatics/16.11.1040 Benson, G. (1999). Tandem repeats finder: a program to analyze DNA sequences. <i>Nucleic Acids Research, 27</i>(2), 573-580. doi:10.1093/nar/27.2.573 Burton, J. N., Adey, A., Patwardhan, R. P., Qiu, R., Kitzman, J. O., & Shendure, J. (2013). Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. <i>Nature Biotechnology, 31</i>(12), 1119-1125. doi:10.1038/nbt.2727 Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. <i>Bioinformatics, 34</i>(17), 884-890. doi:10.1093/bioinformatics/bty560 Hu, J., Fan, J., Sun, Z., & Liu, S. (2020). NextPolish: a fast and efficient genome polishing tool for long-read assembly. <i>Bioinformatics, 36</i>(7), 2253-2255. doi:10.1093/bioinformatics/btz891 Kim, D., Langmead, B., & Salzberg, S. L. (2015). HISAT: a fast spliced aligner with low memory requirements. <i>Nat Methods, 12</i>(4), 357-357. doi:10.1038/nmeth.3317 Langmead, B., & Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. <i>Nat Methods, 9</i>(4), 357-357. doi:10.1038/nmeth.1923 Li, H. (2016). Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. <i>Bioinformatics, 32</i>(14), 2103-2110. doi:10.1093/bioinformatics/btw152 Li, H., & Durbin, R. (2010). Fast and accurate long-read alignment with Burrows-Wheeler transform. <i>Bioinformatics, 26</i>(5), 589-595. doi:10.1093/bioinformatics/btp698 Parra, G., Bradnam, K., & Korf, I. (2007). CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. <i>Bioinformatics, 23</i>(9), 1061-1067. doi:10.1093/bioinformatics/btm071 Pertea, M., Pertea, G. M., Antonescu, C. M., Chang, T.-C., Mendell, J. T., & Salzberg, S. L. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. <i>Nature Biotechnology, 33</i>(3), 290-295. doi:10.1038/nbt.3122 Servant, N., Varoquaux, N., Lajoie, B. R., Viara, E., Chen, C. J., Vert, J. P., . . . Barillot, E. (2015). HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. <i>Genome Biology, 16</i>, 259. doi:10.1186/s13059-015-0831-x Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., & Zdobnov, E. M. (2015). BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. <i>Bioinformatics, 31</i>(19), 3210-3212. doi:10.1093/bioinformatics/btv351 Wang, X., & Wang, L. (2016). GMATA: an integrated software package for genome-scale SSR mining, marker development and viewing. <i>Frontiers in Plant Science, 7</i>, 1350. doi:10.3389/fpls.2016.01350
description: Notes of the
 files: 1Pachypeltis_micranthus.contigs.fasta—The contig sequences of assembled <em>Pachypeltis micranthus </em>genome; 2Pachypeltis_micranthus.cds.fasta—The predicted coding sequences (CDS) of <em>Pachypeltis micranthus.</em> 3Pachypeltis_micranthus.pep.fasta—The predicted protein sequences of <em>Pachypeltis micranthus.</em> 4Pachypeltis_micranthus.chromoses.fasta—The chromosome sequences of <em>Pachypeltis micranthus.</em> 5Pachypeltis_micranthus.genome.gff—The characteristics description of <em>Pachypeltis micranthus </em>genome. 6Pachypeltis_micranthus.repeat.masked.fasta—The annotated tandem repeats (TRs) and transposable elements (TEs) in the <i>P. micranthus</i> genome. 7Pachypeltis_micranthus.repeat.gff—The characteristics description of the tandem repeats (TRs) and transposable elements (TEs) in the <i>P. micranthus</i> genome.