In-house annotated gene set for the pecan weevil, Curculio caryae

This in-house annotated gene set was created using the following methods. RNA was isolated from the head and thorax segments of one adult male and one adult female pecan weevil using the NucleoMag RNA Kit (Macherey-Nagel, Düren, Germany, 744350.1) according to kit protocols. Isolated RNA was processed into PacBio Kinnex sequencing libraries using the Iso-Seq express 2.0 kit (Pacific Biosciences, Menlo Park, CA, USA 103-071-500) and Kinnex full-length RNA kit (Pacific Biosciences, Menlo Park, CA, USA,103-072-000). The prepared library was bound and sequenced at the USDA-ARS Veterinary Pest Genetics Research Unit in Kerrville, Texas, on two Pacific Biosciences SMRT cell trays with a Revio system (Pacific Biosciences, Menlo Park, CA, USA, 102-202-200) beginning with a 2-h pre-extension followed by a 30-h movie collection time. After sequencing, circular consensus sequences from the PacBio Sequel Revio subreads were obtained using the SMRTLink v13.0 software. Reads were subsequently mapped to the repeat-masked genome assembly using minimap2 with arguments for spliced nucleotide sequences (-ax splice:hq) to generate sam mapping files. These were then compressed into bam files using samtools view -bS and used as input for gene model prediction with the Braker version 3.0.8 program (https://github.com/Gaius-Augustus/BRAKER), generating 72,879 gene models. These gene models and amino acid protein predictions were further curated and annotated with gene ontologies and protein domains using InterProScan-5.73-104.0 with PANTHER-19.0 and Pfam-37.2 databases (https://github.com/ebi-pf-team/interproscan), resulting in 19,508 InterProScan results.

本实验室自主构建的注释基因集采用如下实验方法制备。本实验按照试剂盒操作流程，使用NucleoMag RNA试剂盒（Macherey-Nagel，德国迪伦，货号744350.1），从1头成年雄性和1头成年雌性山核桃象鼻虫的头部与胸部组织中分离总RNA。分离得到的RNA采用Iso-Seq Express 2.0建库试剂盒（Pacific Biosciences，美国加利福尼亚州门洛帕克，货号103-071-500）与Kinnex全长RNA建库试剂盒（Pacific Biosciences，美国加利福尼亚州门洛帕克，货号103-072-000）制备为PacBio Kinnex测序文库。制备完成的文库在美国德克萨斯州克尔维尔的USDA-ARS兽医害虫遗传学研究单元，通过搭载Revio测序系统（Pacific Biosciences，美国加利福尼亚州门洛帕克，货号102-202-200）的2张Pacific Biosciences SMRT细胞托盘完成文库结合与测序，测序流程先进行2小时预延伸，随后开展30小时的数据采集。测序完成后，使用SMRTLink v13.0软件从PacBio Sequel Revio的子读段中提取环形一致性序列。随后使用minimap2软件，针对剪接型核苷酸序列设置比对参数（-ax splice:hq），将上述读段比对至经重复序列屏蔽处理的基因组组装结果，生成SAM格式比对文件。再通过samtools view -bS命令将SAM比对文件压缩为BAM格式文件，将其作为输入文件提交至BRAKER 3.0.8版本软件（https://github.com/Gaius-Augustus/BRAKER）进行基因模型预测，最终得到72879个基因模型。随后使用搭载PANTHER-19.0与Pfam-37.2数据库的InterProScan-5.73-104.0软件（https://github.com/ebi-pf-team/interproscan），对上述基因模型及氨基酸蛋白预测结果进行人工校正与功能注释，涵盖基因本体注释与蛋白结构域注释，最终获得19508条InterProScan注释结果。