High-throughput Short-read Sequencing Research Articles

Examining intra-host genetic variation of RSV by short read high-throughput sequencing.

Every viral infection entails an evolving population of viral genomes. High-throughput sequencing technologies can be used to characterize such populations, but to date there are few published examples of such work. In addition, mixed sequencing data are sometimes used to infer properties of infecting genomes without discriminating between genome-derived reads and reads from the much more abundant, in the case of a typical active viral infection, transcripts. Here we apply capture probe-based short read high-throughput sequencing to nasal wash samples taken from a previously described group of adult hematopoietic cell transplant (HCT) recipients naturally infected with respiratory syncytial virus (RSV). We separately analyzed reads from genomes and transcripts for the levels and distribution of genetic variation by calculating per position Shannon entropies. Our analysis reveals a low level of genetic variation within the RSV infections analyzed here, but with interesting differences between genomes and transcripts in 1) average per sample Shannon entropies; 2) the genomic distribution of variation 'hotspots'; and 3) the genomic distribution of hotspots encoding alternative amino acids. In all, our results suggest the importance of separately analyzing reads from genomes and transcripts when interpreting high-throughput sequencing data for insight into intra-host viral genome replication, expression, and evolution.

Utility Analyses of AVITI Sequencing Chemistry.

DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in the genome and transcriptome sequencing of various organisms. Nevertheless, challenges with accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Here, we conduct a comparative analysis using matched DNA and RNA short-reads assays between Element Biosciences' AVITI and Illumina's NextSeq 550 chemistries. Similar comparisons were evaluated for synthetic long-read sequencing for RNA and targeted single-cell transcripts between the AVITI and Illumina's NovaSeq 6000. For both DNA and RNA short-read applications, the study found that the AVITI produced significantly higher per sequence quality scores. For PCR-free DNA libraries, we observed an average 89.7% lower experimentally determined error rate when using the AVITI chemistry, compared to the NextSeq 550. For short-read RNA quantification, AVITI platform had an average of 32.5% lower error rate than that for NextSeq 550. With regards to synthetic long-read mRNA and targeted synthetic long read single cell mRNA sequencing, both platforms' respective chemistries performed comparably in quantification of genes and isoforms. The AVITI displayed a marginally lower error rate for long reads, with fewer chemistry-specific errors and a higher mutation detection rate. These results point to the potential of the AVITI platform as a competitive candidate in high-throughput short read sequencing analyses when juxtaposed with the Illumina NextSeq 550.

PARamrfinder: detecting allele-specific DNA methylation on multicore clusters

The discovery of Allele-Specific Methylation (ASM) is an important research field in biology as it regulates genomic imprinting, which has been identified as the cause of some genetic diseases. Nevertheless, the high computational cost of the bioinformatic tools developed for this purpose prevents their application to large-scale datasets. Hence, much faster tools are required to further progress in this research field. In this work we present PARamrfinder, a parallel tool that applies a statistical model to identify ASM in data from high-throughput short-read bisulfite sequencing. It is based on the state-of-the-art sequential tool amrfinder, which is able to detect ASM at regional level from Bisulfite Sequencing (BS-Seq) experiments in the absence of Single Nucleotide Polymorphism information. PARamrfinder provides the same Allelically Methylated Regions as amrfinder but at significantly reduced runtime thanks to exploiting the compute capabilities of common multicore CPU clusters and MPI RMA operations to attain an efficient dynamic workload balance. As an example, our tool is up to 567 times faster for real data experiments on a cluster with 8 nodes, each one containing two 16-core processors. The source code of PARamrfinder, as well as a reference manual, is available at https://github.com/UDC-GAC/PARamrfinder.

Microbial Community Profiling Protocol with Full-length 16S rRNA Sequences and Emu.

16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species-level resolution. This pipeline provides a low-cost solution for characterizing microbiome composition by exploiting real-time, long-read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full-length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu.

A k-mer-based bulked segregant analysis approach to map seed traits in unphased heterozygous potato genomes.

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.

Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication.

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.

Whole Animal Genome Sequencing: user-friendly, rapid, containerized pipelines for processing, variant discovery, and annotation of short-read whole genome sequencing data.

Advancements in massively parallel short-read sequencing technologies and the associated decreasing costs have led to large and diverse variant discovery efforts across species. However, processing high-throughput short-read sequencing data can be challenging with potential pitfalls and bioinformatics bottlenecks in generating reproducible results. Although a number of pipelines exist that address these challenges, these are often geared toward human or traditional model organism species and can be difficult to configure across institutions. Whole Animal Genome Sequencing (WAGS) is an open-source set of user-friendly, containerized pipelines designed to simplify the process of identifying germline short (SNP and indel) and structural variants (SVs) geared toward the veterinary community but adaptable to any species with a suitable reference genome. We present a description of the pipelines [adapted from the best practices of the Genome Analysis Toolkit (GATK)], along with benchmarking data from both the preprocessing and joint genotyping steps, consistent with a typical user workflow.

Beyond assembly: the increasing flexibility of single-molecule sequencing technology.

The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome assembly and are being used in a variety of ways, including to selectively sequence specific loci with long reads, measure chromatin state and protein-DNA binding in order to investigate the dynamics of gene regulation, and rapidly determine copy number variation. These increasingly flexible uses of single-molecule technologies highlight a young and fast-moving part of the field that is leading to a more accessible era of nucleic acid sequencing.

Phylogenetic Analysis of Allotetraploid Species Using Polarized Genomic Sequences.

Phylogenetic analysis of polyploid hybrid species has long posed a formidable challenge as it requires the ability to distinguish between alleles of different ancestral origins in order to disentangle their individual evolutionary history. This problem has been previously addressed by conceiving phylogenies as reticulate networks, using a two-step phasing strategy that first identifies and segregates homoeologous loci and then, during a second phasing step, assigns each gene copy to one of the subgenomes of an allopolyploid species. Here, we propose an alternative approach, one that preserves the core idea behind phasing-to produce separate nucleotide sequences that capture the reticulate evolutionary history of a polyploid-while vastly simplifying its implementation by reducing a complex multistage procedure to a single phasing step. While most current methods used for phylogenetic reconstruction of polyploid species require sequencing reads to be pre-phased using experimental or computational methods-usually an expensive, complex, and/or time-consuming endeavor-phasing executed using our algorithm is performed directly on the multiple-sequence alignment (MSA), a key change that allows for the simultaneous segregation and sorting of gene copies. We introduce the concept of genomic polarization that, when applied to an allopolyploid species, produces nucleotide sequences that capture the fraction of a polyploid genome that deviates from that of a reference sequence, usually one of the other species present in the MSA. We show that if the reference sequence is one of the parental species, the polarized polyploid sequence has a close resemblance (high pairwise sequence identity) to the second parental species. This knowledge is harnessed to build a new heuristic algorithm where, by replacing the allopolyploid genomic sequence in the MSA by its polarized version, it is possible to identify the phylogenetic position of the polyploid's ancestral parents in an iterative process. The proposed methodology can be used with long-read and short-read high-throughput sequencing data and requires only one representative individual for each species to be included in the phylogenetic analysis. In its current form, it can be used in the analysis of phylogenies containing tetraploid and diploid species. We test the newly developed method extensively using simulated data in order to evaluate its accuracy. We show empirically that the use of polarized genomic sequences allows for the correct identification of both parental species of an allotetraploid with up to 97% certainty in phylogenies with moderate levels of incomplete lineage sorting (ILS) and 87% in phylogenies containing high levels of ILS. We then apply the polarization protocol to reconstruct the reticulate histories of Arabidopsis kamchatica and Arabidopsis suecica, two allopolyploids whose ancestry has been well documented. [Allopolyploidy; Arabidopsis; genomic polarization; homoeologs; incomplete lineage sorting; phasing; polyploid phylogenetics; reticulate evolution.].

De novo transcriptome assemblies of C3 and C4 non-model grass species reveal key differences in leaf development

BackgroundC4 photosynthesis is a mechanism that plants have evolved to reduce the rate of photorespiration during the carbon fixation process. The C4 pathway allows plants to adapt to high temperatures and light while more efficiently using resources, such as water and nitrogen. Despite decades of studies, the evolution of the C4 pathway from a C3 ancestor remains a biological enigma. Interestingly, species with C3-C4 intermediates photosynthesis are usually found closely related to the C4 lineages. Indeed, current models indicate that the assembly of C4 photosynthesis was a gradual process that included the relocalization of photorespiratory enzymes, and the establishment of intermediate photosynthesis subtypes. More than a third of the C4 origins occurred within the grass family (Poaceae). In particular, the Otachyriinae subtribe (Paspaleae tribe) includes 35 American species from C3, C4, and intermediates taxa making it an interesting lineage to answer questions about the evolution of photosynthesis.ResultsTo explore the molecular mechanisms that underpin the evolution of C4 photosynthesis, the transcriptomic dynamics along four different leaf segments, that capture different stages of development, were compared among Otachyriinae non-model species. For this, leaf transcriptomes were sequenced, de novo assembled, and annotated. Gene expression patterns of key pathways along the leaf segments showed distinct differences between photosynthetic subtypes. In addition, genes associated with photorespiration and the C4 cycle were differentially expressed between C4 and C3 species, but their expression patterns were well preserved throughout leaf development.ConclusionsNew, high-confidence, protein-coding leaf transcriptomes were generated using high-throughput short-read sequencing. These transcriptomes expand what is currently known about gene expression in leaves of non-model grass species. We found conserved expression patterns of C4 cycle and photorespiratory genes among C3, intermediate, and C4 species, suggesting a prerequisite for the evolution of C4 photosynthesis. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.

Abundant fungi dominate the complexity of microbial networks in soil of contaminated site: High-precision community analysis by full-length sequencing

During the past decade, the characterization of microbial community in soil of contaminated sites was primarily done by high-throughput short-read amplicon sequencing. However, due to the similarity of 16S rRNA and ITS genes amplicon sequences, the short-read approach often limits the microbial composition analysis at the species level. Here, we simultaneously performed full-length and short-read amplicon sequencing to clarify the community composition and ecological status of different microbial taxa in contaminated soil from a high-resolution perspective. We found that (1) full-length 16S rRNA gene sequencing gave better resolution for bacterial identification at all levels, while there were no significant differences between the two sequencing platforms for fungal identification in some samples. (2) Abundant taxa were vital for microbial co-occurrences network constructed by both full-length and short-read sequencing data, and abundant fungal species such as Mortierella alpine, Fusarium solani, Mrakia frigida, and Chaetomium homopilatum served as the keystone species. (3) Heavy metal correlated with the microbial community significantly, and bacterial community and its abundant taxa were assembled by deterministic process, while the other taxa were dominated by stochastic process. These findings contribute to the understanding of the ecological mechanisms and microbial interactions in site soil ecosystems and demonstrate that full-length sequencing has the potential to provide more details of microbial community.

Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2.

High-throughput short-read sequencing has taken on a central role in research and diagnostics. Hundreds of different assays take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short-read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities and the high capital costs of these technologies have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the rolling circle to concatemeric consensus (R2C2) method to circularize and amplify the short library molecules. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, and regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages.

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara).

High-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing.

Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.

An informatics approach to distinguish RNA modifications in nanopore direct RNA sequencing

Modifications in RNA can influence their structure, function, and stability and play essential roles in gene expression and regulation. Methods to detect RNA modifications rely on biophysical techniques such as chromatography or mass spectrometry, which are low throughput, or on high throughput short-read sequencing techniques based on selectively reactive chemical probes. Recent studies have utilized nanopore-based fourth-generation sequencing methods to detect modifications by directly sequencing RNA in its native state. However, these approaches are based on modification-associated mismatch errors that are liable to be confounded by SNPs. Also, there is a need to generate matched knockout controls for reference, which is laborious. In this work, we introduce an internal comparison strategy termed “IndoC,” where features such as ‘trace’ and ‘current signal intensity’ of potentially modified sites are compared to similar sequence contexts on the same RNA molecule within the sample, alleviating the need for matched knockout controls. We first show that in an IVT model, ‘trace’ is able to distinguish between artificially generated SNPs and true pseudouridine (Ψ) modifications, both of which display highly similar mismatch profiles. We then apply IndoC on yeast and human ribosomal RNA to demonstrate that previously reported Ψ sites show marked changes in their trace and signal intensity profiles compared with their unmodified counterparts in the same dataset. Finally, we perform direct RNA sequencing of RNA containing Ψ intact with a chemical probe adduct (N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide [CMC]) and show that CMC reactivity also induces changes in trace and signal intensity distributions in a Ψ specific manner, allowing their separation from high mismatch sites that display SNP-like behavior.

Application of third-generation sequencing in cancer research.

In the past several years, nanopore sequencing technology from Oxford Nanopore Technologies (ONT) and single-molecule real-time (SMRT) sequencing technology from Pacific BioSciences (PacBio) have become available to researchers and are currently being tested for cancer research. These methods offer many advantages over most widely used high-throughput short-read sequencing approaches and allow the comprehensive analysis of transcriptomes by identifying full-length splice isoforms and several other posttranscriptional events. In addition, these platforms enable structural variation characterization at a previously unparalleled resolution and direct detection of epigenetic marks in native DNA and RNA. Here, we present a comprehensive summary of important applications of these technologies in cancer research, including the identification of complex structure variants, alternatively spliced isoforms, fusion transcript events, and exogenous RNA. Furthermore, we discuss the impact of the newly developed nanopore direct RNA sequencing (RNA-Seq) approach in advancing epitranscriptome research in cancer. Although the unique challenges stillpresent for these new single-molecule long-read methods, they will unravel many aspects of cancer genome complexity in unprecedented ways and present an encouraging outlook for continued application in an increasing number of different cancer research settings.

SCRAMBLER: A Tool for De Novo CRISPR Array Reconstruction and Its Application for Analysis of the Structure of Prokaryotic Populations.

CRISPR arrays are prokaryotic genomic loci consisting of repeat sequences alternating with unique spacers acquired from foreign nucleic acids. As one of the fastest-evolving parts of the genome, CRISPR arrays can be used to differentiate closely related prokaryotic lineages and track individual strains in prokaryotic communities. However, the assembly of full-length CRISPR arrays sequences remains a problem. Here, we developed SCRAMBLER, a tool that includes several pipelines for assembling CRISPR arrays from high-throughput short-read sequencing data. We assessed its performance with model data sets (Escherichia coli strains containing different CRISPR arrays and imitating prokaryotic communities of different complexities) and intestinal microbiomes of extant and extinct pachyderms. Evaluation of SCRAMBLER's performance using model data sets demonstrated its ability to assemble CRISPR arrays correctly from reads containing pairs of spacers, yielding a precision rate of >80% and a recall rate of 60-85% when checked against ground-truth data. Likewise, SCRAMBLER successfully assembled CRISPR arrays from the environmental samples, as attested by their matching with database entries. SCRAMBLER, an open-source software (github.com/biolab-tools/SCRAMBLER), can facilitate analysis of the composition and dynamics of CRISPR arrays in complex communities.

Transcriptome characterization and expression profile of Coix lacryma-jobi L. in response to drought.

Coix lacryma-jobi L. is a very important economic crop widely cultivated in Southeast Asia. Drought affects more than four million square kilometers every year, and is a significant factor limiting agricultural productivity. However, relatively little is known about how Coix lacryma-jobi L. responds to drought treatments. To obtain a detailed and comprehensive understanding of the mechanisms regulating the transcriptional responses of Coix lacryma-jobi L. to drought treatment, we employed high throughput short-read sequencing of cDNA prepared from polyadenylated RNA to explore global gene expression after a seven-day drought treatment. We generated a de novo assembled transcriptome comprising 65,480 unique sequences. Differential expression analysis based on RSEM-estimated transcript abundances identified 5,315 differentially expressed genes (DEGs) when comparing samples from plants following drought-treatment and from the appropriate controls. Among these, the transcripts for 3,460 genes were increased in abundance, whereas 1,855 were decreased. Real-time quantitative PCR for 5 transcripts confirmed the changes identified by RNA-Seq. The results provide a transcriptional overview of the changes in Coix lacryma-jobi L. in response to drought, and will be very useful for studying the function of associated genes and selection of molecular marker of Coix lacryma-jobi L in the future.

Sequencing of methylase-accessible regions in integral circular extrachromosomal DNA reveals differences in chromatin structure

BackgroundAlthough extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In most conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas.MethodsHere, we developed a method of sequencing enzyme-accessible chromatin in circular DNA (CCDA-seq) based on the use of methylase to label open chromatin without fragmentation and exonuclease to enrich ecDNA sequencing depth, followed by long-read nanopore sequencing.ResultsUsing CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding to ecDNA at a single-molecule resolution.ConclusionsThese results deepen the understanding of ecDNA regulatory mechanisms.

Superior resolution characterisation of microbial diversity in anaerobic digesters using full-length 16S rRNA gene amplicon sequencing

In the past decade, the characterisation of the microbial community in anaerobic digestion was primarily done by using high-throughput short-read amplicon sequencing. However, the short-read approach has inherent primer bias and low phylogenetic resolution. Our previous study using Illumina MiSeq suggested that the heterogeneity of AD microbiome was operation-driven. To advance our knowledge towards the complexity of the AD microbiome, we performed full-length 16S rRNA gene amplicon sequencing using PacBio Sequel for a more accurate phylogenetic identification. To this end, purified DNA samples from 19 global anaerobic digesters were sequenced. Sixteen methanogenic archaea were identified at the species level. Among them, Methanosarcina horonobensis and Methanosarcina flavescens had significant presence under specific operating conditions. Methanothrix concilii presented in all digesters sequenced. Unexpectedly, over 90% of the Smithella detected were closely related to alkane-degrading Smithella strains D17 and M82, not Smithella propionica. Using LEfSe and network analysis, the interspecies relationship between the fermentative and syntrophic bacteria was addressed. Comparison of the short- and long-read sequencing results were performed and discussed. From sample preparation to data analysis, this work characterised the digester microbiomes in a superior resolution.