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Long-Read Metagenomics and CAZyme Discovery

Long-Read Metagenomics and CAZyme Discovery

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作者： Alessandra Ferrillo Carl Mathias Kobel Arturo Vera-Ponce de León Sabina Leanti La Rosa Benoit Josef Kunath Phillip Byron Pope Live Heldal Hagen

Microorganisms play a primary role in regulating biogeochemical cycles and are a valuable source of enzymes that have biotechnological applications, such as carbohydrate-active enzymes (CAZymes). However, the inability to culture the majority of microorganisms that exist in natural ecosystems restricts access to potentially novel bacteria and beneficial CAZymes. While commonplace molecular-based culture-independent methods such as metagenomics enable researchers to study microbial communities directly from environmental samples, recent progress in long-read sequencing technologies are advancing the field. We outline key methodological stages that are required as well as describe specific protocols that are currently used for long-read metagenomic projects dedicated to CAZyme discovery. less

关键词： Metagenomics Gene prediction Shotgun Sequencing DNA Extraction Genome Amplification Agarose Gel Electrophoresis Single molecule real time (SMRT) sequencing De-novo Assembly Statistical Calculation DNA Ligation FastP Nanopore Sequencing coding region prediction GenBank Distillation Oryzias latipes Microbial communities Assembly Binning Carbohydrate-active enzymes

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Reconstruction of Archaeal Genomes from Short-Read Metagenomes

Reconstruction of Archaeal Genomes from Short-Read Metagenom...

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作者： Till L. Bornemann Panagiotis S. Adam Alexander J. Probst

As the majority of biological diversity remains unexplored and uncultured, investigating it requires culture-independent approaches. Archaea in particular suffer from a multitude of issues that make their culturing problematic, from them being frequently members of the rare biosphere, to low growth rates, to them thriving under very specific and often extreme environmental and community conditions that are difficult to replicate. OMICs techniques are state of the art approaches that allow direct high-throughput investigations of environmental samples at all levels from nucleic acids to proteins, lipids, and secondary metabolites. Metagenomics, as the foundation for other OMICs techniques, facilitates the identification and functional characterization of the microbial community members and can be combined with other methods to provide insights into the microbial activities, both on the RNA and protein levels. In this chapter, we provide a step-by-step workflow for the recovery of archaeal genomes from metagenomes, starting from raw short-read sequences. This workflow can be applied to recover bacterial genomes as well. less

关键词： Metagenomics Statistical Calculation Bowtie coding region prediction Sequence Editing CRISPR DNA Extraction Microscopy DNA Fragmentation Assay Programming Language Python SNP Calling Genome Annotation Base-calling Prokaryotes Genome curation Short-read sequencing Rare biosphere

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Assembly, Annotation, and Comparative Analysis of Bifidobacterial Genomes

Assembly, Annotation, and Comparative Analysis of Bifidobact...

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作者： Gabriele Andrea Lugli

Genome assembly and annotation are two of the key actions that must be undertaken in order to explore the genomic repertoire of (bifido)bacteria. The gathered information can be employed to genomically characterize a given microorganism, and can also be used to perform comparative genome analysis by including other sequenced (bifido)bacterial strains. Here, we highlight various bioinformatic programs able to manage next generation sequencing data starting from the assembly of a genome to the comparative analyses between strains. less

关键词： Next Generation Sequencing Genomics Phylogenetics Genome Assembly Gene prediction Genome Visualization DNA Sequencing Genome Annotation Database Search coding region prediction Sequence Comparison Statistical Calculation ClustalW Multiple Sequence Alignment Genome Analysis Bifidobacterium Pangenome

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Systematic prediction of functionally linked genes in bacterial and archaeal genomes

Systematic prediction of functionally linked genes in bacter...

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作者： Sergey A. Shmakov Guilhem Faure Kira S. Makarova Yuri I. Wolf Konstantin V. Severinov Eugene V. Koonin

Functionally linked genes in bacterial and archaeal genomes are often organized into operons. However, the composition and architecture of operons are highly variable and frequently differ even among closely related genomes. Therefore, to efficiently extract reliable functional predictions for uncharacterized genes from comparative analyses of the rapidly growing genomic databases, dedicated computational approaches are required. We developed a protocol to systematically and automatically identify genes that are likely to be functionally associated with a ‘bait’ gene or locus by using relevance metrics. Given a set of bait loci and a genomic database defined by the user, this protocol compares the genomic neighborhoods of the baits to identify genes that are likely to be functionally linked to the baits by calculating the abundance of a given gene within and outside the bait neighborhoods and the distance to the bait. We exemplify the performance of the protocol with three test cases, namely, genes linked to CRISPR–Cas systems using the ‘CRISPRicity’ metric, genes associated with archaeal proviruses and genes linked to Argonaute genes in halobacteria. The protocol can be run by users with basic computational skills. The computational cost depends on the sizes of the genomic dataset and the list of reference loci and can vary from one CPU-hour to hundreds of hours on a supercomputer. less

关键词： CRISPR coding region prediction Statistical Calculation NCBI BLAST Python Bioinformatics Genome informatics

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Metagenomics and CAZyme Discovery

Metagenomics and CAZyme Discovery

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作者： Benoit J. Kunath Andreas Bremges Aaron Weimann Alice C. McHardy Phillip B. Pope

Microorganisms play a primary role in regulating biogeochemical cycles and are a valuable source of enzymes that have biotechnological applications, such as carbohydrate-active enzymes (CAZymes). However, the inability to culture the majority of microorganisms that exist in natural ecosystems using common culture-dependent techniques restricts access to potentially novel cellulolytic bacteria and beneficial enzymes. The development of molecular-based culture-independent methods such as metagenomics enables researchers to study microbial communities directly from environmental samples, and presents a platform from which enzymes of interest can be sourced. We outline key methodological stages that are required as well as describe specific protocols that are currently used for metagenomic projects dedicated to CAZyme discovery. less

关键词： Metagenomics Gene prediction DNA Extraction Shotgun Sequencing PCR Genome Amplification De-novo Assembly Statistical Calculation coding region prediction Machine Learning Single molecule real time (SMRT) sequencing Third Generation Sequencing GenBank Microbial communities Assembly Binning Carbohydrate active enzymes

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Creation and Analysis of a Virome: Using CRISPR Spacers

Creation and Analysis of a Virome: Using CRISPR Spacers

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作者： Michelle Davison Devaki Bhaya

Advances in sequencing technology have allowed for the study of complex and previously unexplored microbial and viral populations; however, linking host–phage partners using in silico techniques has been challenging. Here, we describe the flow-through for creation of a virome, and its subsequent analysis with the viral assembly and analysis module “Viritas,” which we have recently developed. This module allows for binning of contigs based on tetranucleotide frequencies, putative phage-host partner identification by CRISPR spacer matching, and identification of ORFs. less

关键词： CRISPR Metagenomics Genome Assembly PCR Gel Electrophoresis coding region prediction Python Bioinformatics Next-generation sequencing Phage annotation

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A Strategy for Identifying Noncoding RNAs Using Whole-Genome Tiling Arrays

A Strategy for Identifying Noncoding RNAs Using Whole-Genome...

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作者： Stephen G. Landt Eduardo Abeliuk

Whole-genome tiling arrays are powerful tools for detecting and characterizing novel RNA transcripts. Here, we describe a complete method combining elements of molecular and computational biology to identify small noncoding RNA (sRNA) transcripts. We focus on the key features of this approach, which include size-fractionation of input RNA, direct detection of array hybridization with antibodies that recognize RNA:DNA hybrids, and correlation-based computational methods for automated sRNA identification and boundary ***-genome tiling arrays are powerful tools for detecting and characterizing novel RNA transcripts. Here, we describe a complete method combining elements of molecular and computational biology to identify small noncoding RNA (sRNA) transcripts. We focus on the key features of this approach, which include size-fractionation of input RNA, direct detection of array hybridization with antibodies that recognize RNA:DNA hybrids, and correlation-based computational methods for automated sRNA identification and boundary determination.

关键词： RNA Extraction PAGE Transillumination Electroelution Tiling Microarray coding region prediction Microarray Transcription Affymetrix Small noncoding RNAs High-density tiling arrays Anti-mouse Anti-rabbit

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Gene Expression Profiling: Metatranscriptomics

Gene Expression Profiling: Metatranscriptomics

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作者： Jack A. Gilbert Margaret Hughes

Metatranscriptomics has been developed to help understand how communities respond to changes in their environment. Metagenomic studies provided a snapshot of the genetic composition of the community at any given time. However, short-timescale studies investigating the response of communities to rapid environmental changes (e.g. pollution events or diurnal light availability) require analysis of changes in the abundance and composition of the active fraction of the community. Metatranscriptomics enables researchers to investigate the actively transcribed ribosomal and messenger RNA from a community. It has been applied to environments as diverse as soil and seawater. This chapter outlines sampling protocols and RNA extraction techniques from these two ecosystems, as well as details a method to enrich mRNA in the extracted nucleic acid. Also, a section is dedicated for outlining a bioinformatic procedure for the analysis of metatranscriptomic *** has been developed to help understand how communities respond to changes in their environment. Metagenomic studies provided a snapshot of the genetic composition of the community at any given time. However, short-timescale studies investigating the response of communities to rapid environmental changes (e.g. pollution events or diurnal light availability) require analysis of changes in the abundance and composition of the active fraction of the community. Metatranscriptomics enables researchers to investigate the actively transcribed ribosomal and messenger RNA from a community. It has been applied to environments as diverse as soil and seawater. This chapter outlines sampling protocols and RNA extraction techniques from these two ecosystems, as well as details a method to enrich mRNA in the extracted nucleic acid. Also, a section is dedicated for outlining a bioinformatic procedure for the analysis of metatranscriptomic datasets.

关键词： Pyrosequencing Gene Expression Profiling RNA Extraction Spectroscopy Picogreen Assay Emulsion PCR coding region prediction PCR Soil Expression Metatranscriptomics Marine

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Obtaining Accurate Translations from Expressed Sequence Tags

Obtaining Accurate Translations from Expressed Sequence Tags

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作者： James Wasmuth Mark Blaxter

The genomes of an increasing number of species are being investigated through the generation of expressed sequence tags (ESTs). However, ESTs are prone to sequencing errors and typically define incomplete transcripts, making downstream annotation difficult. Annotation would be greatly improved with robust polypeptide translations. Many current solutions for EST translation require a large number of full-length gene sequences for training purposes, a resource that is not available for the majority of EST projects. As part of our ongoing EST programs investigating these “neglected” genomes, we have developed a polypeptide prediction pipeline, prot4EST. It incorporates freely available software to produce final translations that are more accurate than those derived from any single method. We describe how this integrated approach goes a long way to overcoming the deficit in training data. less

关键词： coding region prediction NCBI BLAST GenBank Expressed sequence tags ESTs protein translations, simulated transcriptomes

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CGAS

CGAS

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作者： Kwangmin Choi Youngik Yang Sun Kim

Recent advances in genome sequencing technology and algorithms have made it possible to determine the sequence of a whole genome quickly in a cost-effective manner. As a result, there are more than 200 completely sequenced genomes. However, annotation of a genome is still a challenging task. One of the most effective methods to annotate a newly sequenced genome is to compare it with well-annotated and closely related genomes using computational tools and databases. Comparing genomes requires use of a number of computational tools and produces a large amount of output, which should be analyzed by genome annotators. Because of this difficulty, genome projects are mostly carried out at large genome sequencing centers. To alleviate the requirement for expert knowledge in computational tools and databases, we have developed a web-based genome annotation system, called CGAS (a comparative genome annotation system; http://***/CGAS ). This chapter describes how to use CGAS and necessary background knowledge on the computational tools and resources. As an example, a Bacillus subtilis genome is considered as an unannotated target genome and compared with several reference genomes, including Bacillus halodurans, Oceanobacillus iheyensis HTE831, and Bacillus cereus group genomes (representative strain of Bacillus. cereus, Bacillus anthracis). less

关键词： Genome Annotation Sequence Clustering Genomics Gene prediction coding region prediction Multiple Sequence Alignment Structure Analysis PROSITE Protein Structure Analysis Sequence Annotation Genome Sequencing Expasy Sequence Analysis Tools Bacillus subtilis Bacillus halodurans Bacillus cereus ATCC 10987 Bacillus anthracis str. Ames protein domain Bidirectional Best Hit (BBH) genome context

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