- Protocols: Sequencing Library Construction
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- DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.
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Protocols: Sequencing Library Construction
Show next edition. Buy eBook. FAQ Policy. About this book Genome analysis is essential both to understanding the molecular bases of physiological processes and to the development of novel therapies for treating human diseases.
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Back to top. Environ Sci Technol. Microbial diversity within basement fluids of the sediment-buried Juan de Fuca Ridge flank. ISME J. Marine viruses, a genetic reservoir revealed by targeted viromics. Single-cell genomics.
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Nat Biotechnol. Metagenomics - a guide from sampling to data analysis. Microb Inf Exp. Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase. Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.
Multiple displacement amplification compromises quantitative analysis of metagenomes. Nat Methods. Linear amplification for deep sequencing. Nat Protoc.
Peer Reviewed Methods
Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. The effects of variable sample biomass on comparative metagenomics. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. FOCUS: an alignment-free model to identify organisms in metagenomes using non-negative least squares. Comparison of metagenomic samples using sequence signatures. A robust statistical framework for reconstructing genomes from metagenomic data. Cold Spring Harbor Labs Journals.
Tn5 transposase and tagmentation procedures for massively-scaled sequencing projects. Genome Res. Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA. Appl Environ Microbiol. Benjamini Y, Speed TP. Summarizing and correcting the GC content bias in high-throughput sequencing. Nucleic Acids Res. Sources of PCR-induced distortions in high-throughput sequencing datasets.
Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. A quantitative comparison of single-cell whole genome amplification methods. PLoS One. Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection. Lusk RW. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
BMC Biol. Tracking down the sources of experimental contamination in microbiome studies. Ray Meta: scalable de novo metagenome assembly and profiling. MetaVelvet: an extension of Velvet assembler to de novo metagenome assembly from short sequence reads. Bioinform Oxf. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems.
DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.
De novo assembly of human genomes with massively parallel short read sequencing. Sci Rep. QIIME allows analysis of high-throughput community sequencing data. Download references.
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The work conducted by the U. Correspondence to Tanja Woyke. RMB and AC analyzed the metagenomic data. All authors have read and approve this manuscript. Reprints and Permissions. Search all BMC articles Search. Methodology article Open Access Published: 24 October Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community Robert M. Abstract Background The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches.
Results Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. Conclusions This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities. Full size image.
Tagmentation produces variable insert sizes Enzymatic fragmentation based on the Tn5 transposase has been described as a highly efficient library preparation method [ 21 ], with the main obstacle being the control of library insert size. Variation in library GC content may be related to increased PCR cycling at the lowest inputs While PCR enrichment is an often necessary step in the production of sufficient adapter ligated library molecules for sequencing, this step can lead to an artificial, albeit stochastic shift in GC content [ 23 , 24 ].
Taxonomic assignment of reads to references is minimally biased in the Nextera XT and Mondrian libraries down to picogram levels Since this work was performed on a defined mock community, our analyses represent a systematic overview of the factors with the greatest impact on the subsequent metagenomic sequence data.
Comparative metagenomics using community based analyses In addition to the general library statistics and taxonomic read mapping, we also took a community ecology approach to assess the variability between library types and input quantities. Assembly quality varies across library types with potential impact on downstream analyses Assembling reads into larger contiguous fragments is becoming increasingly important in metagenomic studies.
Conclusions The motivation behind our current work was to determine the lower limits of metagenomic library preparation protocols and to assess which library prep performed best down to single picogram DNA input levels.
Methods Description of mock community composition The mock community is composed of 23 bacterial species and 3 archaeal species Additional file 1 : Table S1. Library preparation and sequencing DNA from the same pooled mock community sample was used as input for each of the different library preparation procedures. Analysis of communities using k-tuple frequencies Libraries were also compared using d2Tools, which is a sequence-signature based approach that counts the frequency of k-tuples k2-k10 of each sample, then calculates pairwise dissimilarity matrices using various distance metrics [ 19 ]; Euclidean distances are reported here.
Statistical analyses All statistical analyses and visualizations were performed with R version 3.
References 1. Article Google Scholar 2. Article PubMed Google Scholar 9. Article Google Scholar Acknowledgements The work conducted by the U. Additional information Competing interests The authors declare that they have no competing interests.