Lingen Biotech launched HiFi-meta technology service, a new upgrade of the third-generation metagenomic sequencing assembly solution! Obtain MAGs at the complete map level to facilitate more comprehensive functional gene mining and deepen the relationship between mobile gene units and hosts. We welcome inquiries from users in the field of microecology.
Journal: Gigascience
Impact factor: 7.658
Published: November 2022
1. Research background
The chicken industry is an important industry in my country's animal husbandry. The chicken gut microbiota has a significant impact on the health and growth of the host. Previous research has constructed tens of thousands of chicken gut metagenome assembled genomes (MAG). However, due to limitations of short-read sequencing and assembly technologies, most MAGs are of low quality and contain contaminating reads.
2. Research Methods
Thirty 42-day-old Lingnan yellow broiler chickens were selected, and 150 chyme samples were collected from 5 intestinal compartments of duodenum, jejunum, ileum, cecum and colon for HiFi metagenomic sequencing.
3. Research results
1. HiFi reads can assemble longer contigs
HiFi reads of duodenum, jejunum, ileum, cecum and colon are shown in the table below (Table 1). The N50 length of HiFi reads is about 17 kb. The size of contig N50 preliminarily assembled in the 5 intestinal segments was all above 28kb. In contrast, the N50 size of short-read metagenomes is generally less than 10 kb, suggesting a substantial improvement in contig continuity for HiFi reads. In addition, coverage depth was positively correlated with single-base quality values, indicating that higher coverage depth improves sequence accuracy.
Table 1 HiFi sequencing data statistics
Figure 1 contig assembly statistics
2. Assemble hundreds of complete microbial genomes (MAGs)
27, 40, 62, 285, and 334 medium-quality MAGs were obtained from 5 intestinal segments, respectively (Fig. 3A). 461 and 337 MAGs were assembled at the strain and species levels, respectively, of which 53% and 55% were circular. Ring MAGs also had higher checkM scores (Fig. 3B,C). The study found that higher coverage depth was positively correlated with checkM completeness score, indicating that the improvement of coverage depth is conducive to the assembly of more accurate MAGs.
宏基因组中质粒比宿主基因组更难组装。本研究分别识别出61、67、71、81和78个环状质粒基因组。此外，还鉴定到33、14、14、52和50个环状病毒基因组。
图2 MGAs情况展示
在原核生物中，rRNA和tRNA基因的鉴定是衡量基因组完整性的重要手段。研究者对461个MAGs中的rRNA和tRNA基因进行注释，发现439个(95%)基因组是“RNA完整”的，同时满足rRNA和tRNA标准。大多数MAGs有1到6个rRNA操纵子(图4A)和35到65个tRNA基因拷贝(图4B)。此外，环状基因组中rRNA操纵子和tRNA基因的数量更多。
3.HiFi组装基因组优于短读长组装的MAGs
本研究发现的461个菌株水平MAGs中有384个(83%)是新基因组，包括209个(45%)环状基因组和175个(38%)非环状MAGs(图5A)。与已报道的种水平MAGs相比，本研究的337个种水平基因组中有89个(26%)是新基因组，包括50个(15%)环状基因组和39个(12%)非环状MAGs(图5B)。由于样本量的限制，目前HiFi组装的MAGs比短读长组装的少，但质量明显优于短读长MAGs，基因集的完整性也更高，通过与已发表的两个短读长基因集比较，发现HiFi基因集中有大量的新基因。如图5所示，HiFi组装的微生物基因组平均contig数更少、基因组更长、完整度更高、污染程度更低。
图3 HiFi组装的微生物基因组与短reads组装的MAGs的比较
4.不同肠段的HiFi-MAGs组成存在差异
使用GTDB-Tk将HiFi组装的种水平MAGs与GTDB数据库中的47894个物种簇进行比对、分类学注释。其中，只有1个MAGs被注释为古菌，其余336个MAGs均属于细菌。优势门为厚壁菌门，其次为拟杆菌门和放线菌门，这3个门总共覆盖了317个(94%)MAGs。
前肠由十二指肠、空肠和回肠组成，主要负责饲料的消化和营养物质的吸收。后肠包含盲肠和结肠，它们的功能是发酵、解毒和回收残余的水和盐。前肠和后肠之间的微生物组成有明显的差异。前肠高度富集的5个属:Ligilactobacillus、limmosilactobacillus、乳酸菌、魏斯氏菌和肠球菌。相比之下，后肠的物种多样性更高。因此，对所有肠段进行分析有利于全面了解肠道微生物。
图4 HiFi组装的微生物基因组的系统发育
5.新的基因组表征和新的种属发现
通过GTDB-Tk注释，发现部分MAGs在科、属和种分类学水平上无法进行分类，这表明它们可能是一些新的菌，可能在工业或医学领域有潜在的价值。近年来，乳酸菌及其近亲Ligilactobacillus和Limmosilactobacillus被广泛用作益生菌补充剂，本次组装的2个Ligilactobacillus基因组和1个Limmosilactobacillus基因组还没有注释到种水平，表明这3个基因组可能为益生菌的发展提供了新的基因资源。使用核糖体数据库(RDP)进一步对这些低分类学级别的基因组进行分类，发现这些基因组中有9个和49个可能分别对应于新的属和种，这拓宽了人们对微生物世界的认识。此外，这些新发现的属中约有1 / 3，新发现的种中约有一半没有在短读长MAG数据中发现，这表明它们仅来自HiFi宏基因组数据，进一步显示了HiFi测序在宏基因组研究中的优势。
四、研究结论
HiFi 测序显著提高了宏基因组组装质量，也恢复了以前基于短读长的宏基因组研究中遗漏的大部分新基因组，本研究获得的新基因组和物种将促进肠道微生物组和宿主-微生物群相互作用研究，从而促进家禽资源的可持续发展。
参考文献
Improved microbial genomes and gene catalog of the chicken gut from metagenomic sequencing of high-fidelity long reads.Gigascience.2022