Genome assembly and gene annotations for Glacier lanternfish (Benthosema glaciale)

Here we provide the genome assembly and gene annotations for the Glacier lanternfish (Benthosema glaciale). We provide these for both convenience and because some of the functional annotations of genes/proteins are removed when we prepare these for uploading to ENA. We assembled the species using a pre-release of the EBP-Nor genome assembly pipeline (https://github.com/ebp-nor/GenomeAssembly). HiFiAdapterFilt (Sim et al., 2022) was applied on the HiFi reads to remove possible remnant PacBio adapter sequences. The filtered HiFi reads were assembled using hifiasm (Cheng et al., 2021) with Hi-C integration resulting in a pair of haplotype-resolved assemblies, pseudo-haplotype one (hap1) and pseudo-haplotype two (hap2) for each species. Unique k-mers in each assembly/pseudo-haplotype were identified using meryl (Rhie et al., 2020) and used to create two sets of Hi-C reads, one without any k-mers occurring uniquely in hap1 and the other without k-mers occurring uniquely in hap2. K-mer filtered Hi-C reads were aligned to each scaffolded assembly using BWA-MEM (Li, 2013) with -5SPM options. The alignments were sorted based on name using samtools (Li et al., 2009) before applying samtools fixmate to remove unmapped reads and secondary alignments and to add mate score, and samtools markdup to remove duplicates. The resulting BAM files were used to scaffold the two assemblies using YaHS (Zhou et al., 2022) with default options. FCS-GX (Astashyn et al., 2023) was used to search for contamination. Contaminated sequences were removed. If a contaminant was detected at the start or end of a sequence, the sequence was trimmed using a combination of samtools faidx, bedtools (Quinlan and Hall, 2010) complement, and bedtools getfasta. If the contaminant was internal, it was masked using bedtools maskfasta. The mitochondrion was searched for in contigs and reads using MitoHiFi (Uliano-Silva et al., 2023). The assemblies were manually curated using PretextView. Chromosomes were identified by inspecting the Hi-C contact map in PretextView and named according to homology to kcLamFluv1. Some of the tools used for evaluation have been implemented in the EBP-Nor genome assembly evaluation pipeline (https://github.com/ebp-nor/GenomeEvaluation). We annotated the genome assemblies using a pre-release version of the EBP-Nor genome annotation pipeline (https://github.com/ebp-nor/GenomeAnnotation). First, AGAT (https://zenodo.org/record/7255559) agat_sp_keep_longest_isoform.pl and agat_sp_extract_sequences.pl were used on the GRCz11 genome assembly and annotation to generate one protein (the longest isoform) per gene. Miniprot (Li, 2023) was used to align the proteins to the curated assemblies. UniProtKB/Swiss-Prot (Consortium et al., 2022) release 2022_03 in addition to the Vertebrata part of OrthoDB v11 (Kuznetsov et al., 2022) were also aligned separately to the assemblies. Red (Girgis, 2015) was run via redmask (https://github.com/nextgenusfs/redmask) on the assemblies to mask repetitive areas. GALBA (Brůna et al., 2023; Buchfink et al., 2015; Hoff and Stanke, 2018; Li, 2023; Stanke et al., 2006) was run with the sea lamprey proteins using the miniprot mode on the masked assemblies. The funannotate-runEVM.py script from Funannotate was used to run EvidenceModeler (Haas et al., 2008) on the alignments of sea lamprey proteins, UniProtKB/Swiss-Prot proteins, Vertebrata proteins and the predicted genes from GALBA. The resulting predicted proteins were compared to the protein repeats that Funannotate distributes using DIAMOND blastp, and the predicted genes were filtered based on this comparison using AGAT. The filtered proteins were compared to the UniProtKB/Swiss-Prot release 2022_03 using DIAMOND (Buchfink et al., 2015) blastp to find gene names, and InterProScan was used to discover functional domains. AGATs agat_sp_manage_functional_annotation.pl was used to attach the gene names and functional annotations to the predicted genes.本研究提供了冰川灯笼鱼（Glacier lanternfish，Benthosema glaciale）的基因组组装结果与基因注释信息。我们发布此类数据既为方便科研复用，亦是由于部分基因/蛋白质的功能注释在筹备上传至欧洲核苷酸档案馆（European Nucleotide Archive, ENA）的流程中被移除。 本研究采用EBP-Nor基因组组装流程的预发布版本（https://github.com/ebp-nor/GenomeAssembly）开展该物种的基因组组装。首先借助HiFiAdapterFilt（Sim et al., 2022）对PacBio HiFi测序读段（reads）进行过滤，去除可能残留的PacBio接头序列。随后使用hifiasm（Cheng et al., 2021）对过滤后的HiFi读段进行组装，并整合Hi-C数据，最终获得两套单倍型解析的组装结果：伪单倍型1（hap1）与伪单倍型2（hap2）。 利用meryl（Rhie et al., 2020）分别识别两套组装结果/伪单倍型中的独特k-mer，据此生成两组Hi-C读段：一组移除所有仅在hap1中出现的k-mer，另一组移除所有仅在hap2中出现的k-mer。将经k-mer过滤的Hi-C读段以BWA-MEM（Li, 2013）的-5SPM参数比对至各自的骨架组装基因组。使用samtools（Li et al., 2009）按名称比对结果进行排序，随后执行samtools fixmate以移除未比对读段与二级比对结果，并添加mate评分；再通过samtools markdup去除重复比对读段。最终得到的BAM文件将用于通过YaHS（Zhou et al., 2022）的默认参数对两套组装结果进行骨架化操作。 使用FCS-GX（Astashyn et al., 2023）对组装序列开展污染筛查，移除检测到的污染序列。若污染序列位于某条序列的起始或末端，则通过samtools faidx、bedtools（Quinlan and Hall, 2010）complement与bedtools getfasta的组合操作对该序列进行修剪；若污染序列位于序列内部，则使用bedtools maskfasta对其进行遮蔽。借助MitoHiFi（Uliano-Silva et al., 2023）在重叠群（contig）与测序读段中搜索线粒体基因组。通过PretextView对组装结果进行人工手动校正。通过在PretextView中检视Hi-C接触图谱识别染色体，并依据与kcLamFluv1的同源性完成命名。本研究使用的部分评估工具已集成于EBP-Nor基因组组装评估流程（https://github.com/ebp-nor/GenomeEvaluation）中。 本研究采用EBP-Nor基因组注释流程的预发布版本（https://github.com/ebp-nor/GenomeAnnotation）对基因组组装结果进行注释。首先，针对GRCz11基因组组装与注释文件，使用AGAT（https://zenodo.org/record/7255559）的agat_sp_keep_longest_isoform.pl与agat_sp_extract_sequences.pl工具，为每个基因生成一条最长的蛋白亚型（isoform）。使用Miniprot（Li, 2023）将生成的蛋白序列比对至经人工校正的组装基因组。此外，分别将UniProtKB/Swiss-Prot（Consortium et al., 2022）2022_03版本数据库以及OrthoDB v11的脊椎动物（Vertebrata）子集（Kuznetsov et al., 2022）的序列比对至组装基因组。通过redmask（https://github.com/nextgenusfs/redmask）调用Red（Girgis, 2015）对组装基因组的重复区域进行软遮蔽。使用GALBA（Brůna et al., 2023; Buchfink et al., 2015; Hoff and Stanke, 2018; Li, 2023; Stanke et al., 2006）的miniprot模式，以七鳃鳗蛋白作为参考对遮蔽后的组装基因组进行基因预测。使用Funannotate中的funannotate-runEVM.py脚本，对七鳃鳗蛋白、UniProtKB/Swiss-Prot蛋白、脊椎动物蛋白的比对结果以及GALBA预测的基因运行EvidenceModeler（Haas et al., 2008）。将得到的预测蛋白序列与Funannotate内置的蛋白重复序列库通过DIAMOND blastp进行比对，随后使用AGAT基于该比对结果对预测基因进行过滤。将过滤后的蛋白序列与UniProtKB/Swiss-Prot 2022_03版本数据库通过DIAMOND（Buchfink et al., 2015）blastp进行比对以获取基因名称，并使用InterProScan预测功能结构域。使用AGAT的agat_sp_manage_functional_annotation.pl工具将基因名称与功能注释信息关联至预测基因。