Genomes and annotations of 4 Diamesa species

Here we provide the genome assemblies and genome annotations of four Diamesa species (D. hyperborea, D. lindrothi, D. serratosioi, D. tonsa). We assembled the species using a pre-release of the EBP-Nor genome assembly pipeline (https://github.com/ebp-nor/GenomeAssembly). KMC (Kokot et al. 2017) was used to count k-mers of size 32 in the PacBio HiFi reads, excluding k-mers occurring more than 10,000 times. Genomescope (Ranallo-Benavidez et al. 2022) was run on the k-mer histogram output from KMC to estimate genome size, heterozygosity and repetitiveness while ploidy level was investigated using Smudgeplot (Ranallo-Benavidez et al. 2022). 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). 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. 2023) with default options. FCS-GX (Astashyn et al. 2024) was used to search for contamination. Contaminated sequences were removed. The mitochondrion was searched for in reads using Oatk (Zhou et al. 2024). 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) scripts agat_sp_keep_longest_isoform.pl and agat_sp_extract_sequences.pl were used on the fruit fly (Drosophila melanogaster) genome assembly (BDGP6.46 (GCA_000001215.4) from Ensembl) 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 (Coudert et al. 2023) release 2022_03 in addition to the arthropoda part of OrthoDB v11 (Kuznetsov et al. 2023) 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. In addition, we ran Earl Grey (Baril et al. 2024) to annotate transposable elements. GALBA (Brunå et al. 2023, Li 2023, Buchfink et al. 2015, Hoff & Stanke 2019, Stanke et al. 2006) was run with the fruit fly 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 the fruit fly proteins, UniProtKB/Swiss-Prot proteins, arthropoda 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4种直突摇蚊属（Diamesa）物种（D. hyperborea、D. lindrothi、D. serratosioi、D. tonsa）的基因组组装（genome assemblies）结果与基因组注释（genome annotations）信息。 本研究使用EBP-Nor基因组组装流程（genome assembly pipeline）的预发布版本对上述物种进行基因组组装，相关流程代码托管于https://github.com/ebp-nor/GenomeAssembly。使用KMC（Kokot等人，2017）统计PacBio HiFi测序reads中长度为32的k聚体（k-mer）频率，并过滤掉出现次数超过10000次的k聚体。基于KMC输出的k聚体频率直方图运行Genomescope（Ranallo-Benavidez等人，2022），以估算基因组大小、杂合度与重复序列占比；同时使用Smudgeplot（Ranallo-Benavidez等人，2022）分析基因组倍性水平。使用HifiAdapterFilt（Sim等人，2022）处理HiFi测序reads，以去除残留的PacBio接头序列。 使用hifiasm（Cheng等人，2021）结合Hi-C测序数据对过滤后的HiFi reads进行组装，得到两套单倍型解析的基因组组装结果，即假单倍体1（hap1）与假单倍体2（hap2）。使用meryl（Rhie等人，2020）识别每套组装结果/假单倍体中的特异性k聚体，据此构建两组Hi-C reads：一组不含仅在hap1中出现的特异性k聚体，另一组不含仅在hap2中出现的特异性k聚体。 使用BWA-MEM（Li，2013）并指定-5SPM参数，将经过k聚体过滤的Hi-C reads比对至每套支架化组装（scaffolded assembly）结果。使用samtools（Li等人，2009）按reads名称对比对结果进行排序，随后依次运行samtools fixmate以去除未比对reads、二级比对reads并添加配对得分，以及samtools markdup以去除重复reads。使用默认参数的YaHS（Zhou等人，2023），基于上述处理得到的BAM文件对两套组装结果进行支架化锚定。使用FCS-GX（Astashyn等人，2024）检测基因组组装结果中的污染序列，并移除污染片段。使用Oatk（Zhou等人，2024）在测序reads中检索线粒体序列。 本研究使用EBP-Nor基因组注释流程（genome annotation pipeline）的预发布版本对上述基因组组装结果进行注释，相关流程代码托管于https://github.com/ebp-nor/GenomeAnnotation。首先，基于果蝇（Drosophila melanogaster）的基因组组装结果（Ensembl数据库中的BDGP6.46版本，登录号GCA_000001215.4）与注释信息，使用AGAT（https://zenodo.org/record/7255559）的两个脚本agat_sp_keep_longest_isoform.pl与agat_sp_extract_sequences.pl，为每个基因生成一条最长可变剪接体对应的蛋白序列。使用Miniprot（Li，2023）将上述果蝇蛋白序列比对至经过整理的基因组组装结果。此外，分别将UniProtKB/Swiss-Prot数据库2022_03版本（Coudert等人，2023）以及OrthoDB v11数据库的节肢动物亚库（Kuznetsov等人，2023）的蛋白序列比对至基因组组装结果。通过redmask（https://github.com/nextgenusfs/redmask）运行Red（Girgis，2015）对基因组组装结果进行重复序列屏蔽。此外，使用Earl Grey（Baril等人，2024）注释基因组中的转座因子。 使用GALBA（Brunå等人，2023；Li，2023；Buchfink等人，2015；Hoff & Stanke，2019；Stanke等人，2006），以Miniprot比对模式结合果蝇蛋白序列，对经过重复序列屏蔽的基因组组装结果进行基因预测。使用Funannotate工具包中的funannotate-runEVM.py脚本，基于果蝇蛋白、UniProtKB/Swiss-Prot蛋白、节肢动物蛋白的比对结果以及GALBA预测的基因，运行EvidenceModeler（Haas等人，2008）进行整合基因预测。使用DIAMOND的blastp工具，将整合预测得到的蛋白序列与Funannotate内置的蛋白重复序列数据库进行比对，随后通过AGAT基于比对结果过滤预测基因。使用DIAMOND（Buchfink等人，2015）的blastp工具，将过滤后的蛋白序列与UniProtKB/Swiss-Prot 2022_03版本数据库进行比对以获取基因名称；同时使用InterProScan预测蛋白功能结构域。使用AGAT的agat_sp_manage_functional_annotation.pl脚本，将基因名称与功能注释信息关联至预测得到的基因模型。