Restriction Endonucleases

Robinson, Derek; Walsh, Paul R.; Bonventre, J. V.

doi:10.1002/0471223905.ch9

Cited by 4 publications

(2 citation statements)

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“…Star sites are associated with a canonical recognition site, but usually differ by one or more bases. This results in non-specific fragments which presence was not considered in the in silico prediction [48]. Initial DNA quality is also a crucial determinant for sequencing reduced representative fractions of the genome.…”

Section: Discussionmentioning

confidence: 99%

Optimized In Vitro Restriction Digestion Protocol for Preparing Maize and Barley ddRAD-Seq Libraries

Puchta-Jasińska,

Bolc,

Piechota

et al. 2023

Agronomy

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In recent years, high-throughput sequencing methods have become increasingly popular in molecular biology laboratories, mainly due to the relatively low cost of small, benchtop platforms, the simplicity of library preparation, and the low price per unit of information. Sequencing huge and complex genomes, such as cereal genomes, remains challenging and may not always be necessary. Therefore, several techniques have been developed to sequence a reduced representation of the genome. The most flexible and widely used of these is ddRAD-Seq, which uses a pair of restriction enzymes to generate a pool of DNA fragments. The aim of this study was to validate in vitro the efficacy of different combinations of restriction enzymes for ddRAD-Seq library construction in barley and maize. Eleven pairs of restriction enzymes were selected and tested to determine the concentrations of fragments with the expected length range and to select suitable pairs for sampling the genomes of these two cereals using ddRAD-Seq. For the selected pairs, i.e., PstI—MspI and HindIII—FspBI for barley and maize, respectively, libraries were prepared for NGS sequencing on Illumina MiSeq. Sequencing confirmed the suitability of the selected enzymes to perform ddRAD-Seq in different genotypes. The results presented can be used for extensive research on these important cereal species.

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Section: Discussionmentioning

confidence: 99%

Optimized In Vitro Restriction Digestion Protocol for Preparing Maize and Barley ddRAD-Seq Libraries

Puchta-Jasińska,

Bolc,

Piechota

et al. 2023

Agronomy

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“…Therefore, usage of methylation-sensitive restriction endonucleases should be very careful in RiboFR-Seq. For example, XbaI could not cleave the genomic DNA of E. coli GM2163 with deleted dam and dcm at recognition sequences TCTAGATC and GATCTAGA ( 58 ). As more and more restriction enzymes have been discovered and commercially available, RiboFR-Seq would be suitable for broad applications in further metagenomic studies.…”

Section: Discussionmentioning

confidence: 99%

RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

Zhang

Wang

et al. 2016

Nucleic Acids Res

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Abstract16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities.

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Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection

et al. 2019

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Restriction Endonucleases

Cited by 4 publications

References 0 publications

Optimized In Vitro Restriction Digestion Protocol for Preparing Maize and Barley ddRAD-Seq Libraries

Optimized In Vitro Restriction Digestion Protocol for Preparing Maize and Barley ddRAD-Seq Libraries

RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection

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