With the continuous development of sequencing technology, omics research has become more in-depth. In the field of microecological research, the amplicon sequencing technology limited by the read length of the second-generation sequencing ushered in the era of "liberated" comprehensive development and optimization of the third-generation sequencing-the advantages of PacBio's full-length amplicon sequencing gradually emerged: With the advantages of ultra-long read length, high accuracy, and high resolution , the full-length amplicon sequencing technology has played an important role in environmental protection, medical diagnosis, animal husbandry, sewage treatment and other fields. Here, I would like to share with you a number of full-length amplicon research applications based on the PacBio third-generation sequencing platform, and let us have an in-depth understanding of the new methods and new ideas provided by the third-generation sequencing technology for microecological research!
01. Research on environmental bacteria
◆ Literature title: Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing
◆ Journal: Microbiome
◆ Release time: January 2021
◆ Content summary:
Freshwater ecosystems are home to cosmopolitan phytoplankton bacterial lineages despite their discrete habitats. Lineage divisions based on greater than 97% similarity in 16S rRNA gene sequences remain unresolved within bacterial lineage diversity and phylogeography, which are key to understanding the ubiquitous ecological evolutionary processes underlying these lineages. In this study, long-read amplicon sequencing of the nearly full-length 16S rRNA gene and adjacent internal ribosomal transcribed spacer sequences was performed to reveal intralineage diversity of pelagic bacterioplankton assemblages in 11 deep-water lakes in Japan and Europe . Analysis by single nucleotide calls, validated by shotgun metagenomic sequencing, identified 7-10 amplicon sequence variants for each of 11 major bacterial lineages and suggested common Domain, heterogeneous and temporal microdiversity. Clusters of samples with similar within-lineage population composition were identified, which has consistently supported genetic isolation between Japan and Europe. At regional scales (up to hundreds of kilometers), dispersal between lakes is unlikely to be the limiting factor, whereas environmental factors or genetic drift are potential determinants of population composition. Different lineages showed varying degrees of microdiversity, suggesting highly diverse lineages, with the lowest degree of intra-lineage diversity being the dominant secondary species lineage (CL500-11), suggesting their unrestricted inter-lake dispersal, Although the thermosphere is a more isolated habitat than the thermosphere.
◆ Research highlights:
The new approach utilizing long-length amplicon sequencing complements the limited resolution of short-read amplicon sequencing and the limited sensitivity of metagenomic assembly-based approaches, highlighting complex ecological processes that are prevalent in freshwater phytoplankton bacterial lineages.
Figure 1. Comparison of single nucleotide polymorphisms (SNPs) based on amplified sequence variation (ASV) in Biwako CL500-11 sequence and SNPs detected by metagenomic technology
02. Research on environmental fungi
◆ Literature title: Long- and short-read metabarcoding technologies reveal similar spatio-temporal structures infectious communities
◆ Journal: Molecular Ecology Resources
◆ Release time: March 2021
◆ Content summary:
Fungal communities play an important role in terrestrial ecosystems, but it is still quite challenging to identify fungal species from the environment and conduct phylogenetic positioning studies. In this study, the researchers used four sequencing strategies to study the spatio-temporal structure of the fungal community: the ITS2 region (~300-400bp long) amplicon was sequenced using the illumina Miseq, Ion Torrent IonS5 and Pacbio RSII platforms, and the PacbioRSII platform was used to sequence the amplicons. Amplicon sequencing of the full length of ITS and partial region of LSU (1200-1600bp in length) was carried out. The study found. The resulting community structure and diversity depend more on statistical methods than on sequencing technologies and platforms. Using long-length reads can construct a phylogenetic tree from meta-reads, which is helpful for the classification and identification of sequences; but at the same time, there are problems with different community denoising algorithms when the length is selected. In this regard, the researchers proposed a solution, which is to divide the long reads into short homologous regions and denoise them, and then reconstruct the complete denoised reads. In terms of research program selection, the researchers recommend a mixed approach: short reads to achieve high sampling quantity and depth, and long reads to characterize local species pools to improve species identification and phylogenetic analysis.
◆ Research highlights:
Comparing the difference of different sequencing platforms, sequencing schemes and analysis methods on the microecological characterization of samples, a mixed analysis scheme of long read and short read was proposed to optimize the study of the spatiotemporal structure of fungal microecology.
Figure 2. a) Fungal ITS amplified region overview; b) Total number of DADA2 ASVs and proportion of demultiplexed reads successfully mapped to ASVs using LSUx from different rDNA regions extracted from a set of long PacBio amplicon sequences
03. Research on animal microecology
◆ 文献标题：Using PacBio sequencing to investigatethe effects of treatment with lactic acid bacteria or antibiotics on cowendometritis
使用PacBio测序研究乳酸菌和抗生素治疗奶牛子宫内膜炎的效果
◆ 期刊：Electronic Journal of Biotechnology
◆ 发表时间：2021年3月
◆ 内容摘要：
子宫内膜炎是奶牛最常见的疾病，一般使用抗生素进行治疗。乳酸菌既能抑制病原体生长，又有治疗子宫内膜炎的潜力。本研究采用PacBio单分子实时测序技术，对31例子宫内膜炎奶牛子宫粘液样本中微生物群的16S rRNA基因全长进行了测序，其中乳酸菌处理组(实验组)和抗生素对照组(对照组)分别为实验组(E组)和对照组(C组)。比较治疗前后采集的微生物群。结果：两种处理后细菌物种丰富度均显著高于处理前，但细菌多样性差异无显著性。经乳酸菌和抗生素处理后，Lactobacillus helveticus,Lactococcus lactis,Lactococcus raffinolactis,Pseudomonas alcaligenes和Pseudomonasveronii等菌种丰度增加。数量显着减少的细菌种类因奶牛是否使用了乳酸菌或抗生素而有所不同。Staphylococcus equorum和Treponemabrennaborense的丰度在乳酸菌处理后增加，在抗生素处理后降低。根据基于COG的功能宏基因组预测，384个功能蛋白在治疗后有显著差异。E组和C组蛋白表达途径均明显高于治疗前(p<0.05)。
◆ 结论：
本研究发现乳酸菌可用于治疗子宫内膜炎，恢复正常生理状态，同时避免了抗生素治疗带来的有益微生物群丰度降低的缺点，表明乳酸菌具有替代抗生素用于治疗牛子宫内膜炎的潜力。
图3. 不同特定COG注释的代谢途径和相应蛋白质的数量
04、昆虫微生态研究
◆ 文献标题：Differences in the intenstinalmicrobiota between insecticide-resistant and -sensitive Aedes albopictus basedon full-length 16S rRNA sequencing.
基于16S rRNA全长测序研究抗杀虫剂及对杀虫剂敏感的白纹伊蚊的肠道微生态差异。
◆ 期刊：Microbiology Open
◆ 发表时间：2021年2月
◆ 内容摘要：
白纹伊蚊的肠道共生菌在宿主对杀虫剂的抗性中发挥潜在作用。在这项研究中，利用16s rRNA全长测序技术，对具有杀虫剂抗性和敏感性白纹伊蚊肠道微生态进行了测序，并分析了两者之间肠道微生物群落的差异。此外，研究在好氧和厌氧环境中使用六种培养基培养和分析共生菌。我们发现两种白纹伊蚊的肠道微生物群的多样性和丰度存在显着差异。发现体外培养的共生菌主要是兼性厌氧菌。米沙雷菌（Serratia oryzae）、朱氏不动杆菌（Acinetobacter junii）等培养菌可能具有促进杀虫剂抗药性发展的作用。研究表明肠道微生态有助于增强白纹伊蚊对杀虫剂的抗性，并指出16s rRNA全长测序对研究蚊子的肠道微生态有分析优势。
◆ 研究亮点：
研究采用16s rDNA全长测序技术对具有杀虫剂抗性和不具杀虫剂抗性的白纹伊蚊的肠道微生态进行测序，基于全长测序的高分辨率的识别出白纹伊蚊肠道微生态中对抗杀虫剂有积极响应的沙雷氏菌和不动杆菌物种，说明16s全长测序可作为研究蚊虫肠道微生态的理想技术。
图4.抗药性和敏感型白纹伊蚊肠道微生态的多样性指数差异。a-c分别为Simpson指数、Shannon指数和Chao指数。
05、人体微生态研究
◆ 文献标题：High-Resolution Differentiation ofEnteric Bacteria in Premature Infant Fecal Microbiomes Using a Novel rRNAAmplicon
利用一种新的rRNA扩增子高分辨率鉴定早产儿粪便微生物组的肠道细菌
◆ 期刊：mBio
◆ 发表时间：2021年2月
◆ 内容摘要：
随着微生物群的进化，识别和跟踪微生物菌株是微生物组研究领域的主要挑战。研究者利用了一种新的测序试剂盒，它结合了DNA提取、rRNA操纵子大区域的PCR扩增和下游生物信息学数据分析。来自两个不同新生儿重症监护病房(NICU)的共入院双胞胎的纵向微生物组样本被用跨越16S和23S rRNA基因并比对到新的自定义的16S-23S rRNA数据库的2500个碱基扩增子进行了分析。使用DADA2推断的扩增序列变体(ASV)为区分rRNA变体提供了足够的分辨率，这些菌株与克雷伯氏菌、大肠杆菌和肠杆菌菌株关系密切，但之前未测序，这些菌株是NICU入院后在这些婴儿肠道中定殖的第一批细菌。不同的ASV群体随着时间的推移在双胞胎之间被监测，证明了追踪共生体和病原体的来源和传播的可能性。从长扩增子测序获得的高分辨率分类，可在医院环境中婴儿建立微生物组，在时间和空间上跟踪菌株。
◆ 研究亮点：
本研究突破16s rDNA扩增子全长的局限，充分利用PacBio三代测序的技术优势，将条形码范围扩大到16s+23s的超长区域，在人体微生态研究中实现了菌株级别分辨，显著提高了扩增子研究的分辨率。
图5. 双胞胎A/B和Y/Z的细菌群落构成。研究对两对双胞胎在出生后第1周至第8周(双胞胎A/B)或第9周(双胞胎Y/Z)的粪便样本进行了群落结构组成分析。
从上述5篇近期发表的扩增子研究进展中不难看出，伴随着三代测序技术应用的推广和深入研究者已经不能满足基于短读取的扩增子研究，而更倾向于打破二代测序读长短的瓶颈，将三代的全长扩增子研究应用到更多领域研究，如真菌群落，真核生物群落研究等；并将全长扩增子的范围进一步扩大，不仅仅局限在既往研究的指定经典区域，而将扩增子区域选择范围放得更大（如16s+23S, ITS+LSU等），获得更长的序列信息以换取更高的物种分辨率。
以细菌的16S研究为例，基于16s全长区域，可以通过长度长测序获得更长的序列信息和物种变异位点，在此前提下长读取研究方案可以在同一分类水平下实现更多物种分类，更有研究者基于16S全长测序获得的长序列中的单核苷酸变异信息实现了对菌株的区分，获得详尽的菌株分类信息，在更精细的物种鉴定中显示出绝对优势这一点很大程度满足了微生物群落研究者对物种鉴定精细度的要求。
由于16S全长测序获取的长序列信息，可极大程度提升和改善序列不能被识别的状况。有研究者比对了基于16s不同高变区和16s全长研究的差异：不难看出V1-V9区引物很少有序列不能被明确分类，相比之下，仅仅开展V4区的研究，则有极高比例的序列无法实现明确分类。
此外，通过聚类OTU，并与模拟物种库进行比较，研究者还发现，V1-V9区研究实现了对样本的最佳还原（下图），物种的识别和定量是最接近模拟物种库原貌的，这一点说明了基于三代平台的16s全长测序在保真效果上的巨大优势。
同时，由于三代测序技术在建库等实验操作过程中无需PCR过程，这就最大程度保持了样本的真实微生态状态，降低了研究结果偏差。
不难看出，伴随着三代长度长测序技术的发展，全长扩增子测序的优势逐渐显现，高分辨率表征微生态群落结构，精细的物种识别和对样本状态的高保证还原。全场扩增子测序技术势必取代基于NGS技术的扩增子研究，成为微生态研究领域的新热点和发展的必然趋势。