MetaChick: characterization of the chicken caecal metagenome by deep shotgun sequencing

Data sourcesThis dataset was constructed using the samples of the MetaChick project (phase 1) corresponding to the cecal content of 340 animals. Sequencing data and associated metadata have been submitted to INSDC (bioproject: PRJEB38174). Sequencing data QC and metagenomic assemblyFirst, sequencing adapters removal and read trimming was performed with fastxtend. Reads mapped on the host genome (GRCg7b GCA_016699485.1) with bowtie2 were removed with samtools. Finally, metagenomic assembly was performed with metaSPAdes v3.14.1. Contigs of less than 1500 bp were removed. MAGs recoveryMAGs were generated with MetaBAT 2 (multi-coverage mode) and MAGs quality was assessed with CheckM. MAGs with completeness < 70% or contamination > 5% or N50 < 8Kb were discarded. Pairwise Average Nucleotide Identity (ANI) was computed for all recovered MAGs with fastANI and dereplication at species level (ANI cutoff = 95%). Non-redundant gene catalogGenes were predicted on all contigs from metagenomic assemblies with Prodigal (parameters : -m -p meta). Genes were pooled and clustered with cd-hit-est (parameters -c 0.95 -aS 0.90 -G 0 -d 0 -M 0 -T 0) by choosing those from the longest contigs as representatives. MSPs recoveryA raw gene abundance table (13,6M genes quantified in 340 samples) was generated with meteorMeteor. Then, co-abundant genes were binned in Metagenomic Species Pan-genomes (MSPs, i.e. gene clusters that likely belong to the same microbial species) using MSPminer. MAGs and MSPs taxonomic annotationDereplicated MAGs were annotated with GTDB-Tk based on GTDB r214. Then, MAGs taxonomic annotation was propagated to the corresponding MSPs. Construction of the phylogenetic tree39 universal phylogenetic markers genes were extracted from the dereplicated MAGs with fetchMGs. Then, the markers were separately aligned with MUSCLE. The 40 alignments were merged and trimmed with trimAl (parameters: -automated1). Finally, the phylogenetic tree was computed with FastTreeMP (parameters: -gamma -pseudo -spr -mlacc 3 -slownni). Mapping rate distribution across public cohortsWe generated mapping rate distribution plots using Meteor2 (default parameters), comparing performance between: PRJEB38174 (cohort used in catalogue assembly) and PRJEB29033, PRJEB33338, PRJEB53667 (independent cohort not used in assembly).数据集来源 本数据集基于MetaChick项目（第一阶段）的340份动物盲肠内容物样本构建。测序数据及相关元数据已提交至国际核苷酸序列数据库协作组织（INSDC，International Nucleotide Sequence Database Collaboration），生物项目编号为PRJEB38174。 测序数据质控与宏基因组组装 首先，使用fastxtend完成测序接头去除与读段修剪；利用bowtie2比对至宿主基因组（GRCg7b GCA_016699485.1）的读段将通过samtools过滤移除；最终使用metaSPAdes v3.14.1进行宏基因组组装，并剔除长度小于1500 bp的重叠群。 宏基因组组装基因组（MAGs，Metagenome-Assembled Genomes）提取 使用MetaBAT 2的多覆盖模式生成MAGs，并通过CheckM评估MAGs质量。剔除完整度低于70%、污染率高于5%或N50小于8 kb的MAGs。使用fastANI计算所有回收得到的MAGs的平均核苷酸一致性（ANI，Average Nucleotide Identity），并以95%的ANI阈值进行物种级去冗余。 非冗余基因集构建 利用Prodigal（参数：-m -p meta）对所有宏基因组组装得到的重叠群进行基因预测；将预测得到的所有基因合并后，使用cd-hit-est（参数：-c 0.95 -aS 0.90 -G 0 -d 0 -M 0 -T 0）进行聚类，并以最长重叠群对应的基因作为代表序列。 宏基因组物种泛基因组（MSPs，Metagenomic Species Pan-genomes）提取 利用Meteor生成原始基因丰度表（在340份样本中定量得到1360万条基因）；随后使用MSPminer将共丰度基因聚类为MSPs，即归属同一微生物物种的基因簇集合。 MAGs与MSPs的分类注释 基于基因组分类数据库（GTDB，Genome Taxonomy Database）r214版本，使用GTDB-Tk对去冗余后的MAGs进行分类注释；随后将MAGs的分类注释信息传递至对应的MSPs。 系统发育树构建 利用fetchMGs从去冗余后的MAGs中提取39个通用系统发育标记基因；使用MUSCLE分别对各标记基因进行序列比对；将40条比对结果合并后，使用trimAl（参数：-automated1）进行修剪；最终使用FastTreeMP（参数：-gamma -pseudo -spr -mlacc 3 -slownni）计算得到系统发育树。 公共队列的比对率分布分析 利用Meteor2（默认参数）生成比对率分布图谱，对比本研究用于构建基因集的队列PRJEB38174，与未用于基因集构建的独立队列PRJEB29033、PRJEB33338、PRJEB53667的测序数据比对性能。