16 results on '"Nextera XT"'
Search Results
2. The efficiency of Nextera XT tagmentation depends on G and C bases in the binding motif leading to uneven coverage in bacterial species with low and neutral GC-content.
- Author
-
Segerman, Bo, Ástvaldsson, Ásgeir, Mustafa, Linda, Skarin, Joakim, and Skarin, Hanna
- Subjects
CAMPYLOBACTER jejuni ,SPECIES ,SALMONELLA enterica ,MYCOBACTERIUM tuberculosis ,NUCLEOTIDE sequencing ,LISTERIA monocytogenes - Abstract
Whole-genome sequencing (WGS) is becoming the new standard for bacterial high-resolution typing and the performance of laboratories is being evaluated in interlaboratory comparisons. The use of the Illumina Nextera XT library preparation kit has been found to be associated with poorer performance due to a GC-content-dependent coverage bias. The bias is especially strong when sequencing low GC-content species. Here, we have made an in-depth analysis of the Nextera XT coverage bias problem using data from a proficiency test of the low GC-content species Campylobacter jejuni. We have compared Nextera XT with Nextera Flex/DNA Prep and examined the consequences on downstream WGS analysis when using different quantities of raw data. We have also analyzed how the coverage bias relates to differential usage of tagmentation cleavage sites. We found that the tagmentation site was characterized by a symmetrical motif with a central AT-rich region surrounded by Gs and Cs. The Gs and Cs appeared to be the main determinant for cleavage efficiency and the genomic regions that were associated with low coverage only contained low-efficiency cleavage sites. This explains why low GC-content genomes and regions are more subjected to coverage bias. We furthermore extended our analysis to other datasets representing other bacterial species. We visualized how the coverage bias was large in low GC-content species such as C. jejuni, C. coli, Staphylococcus aureus, and Listeria monocytogenes, whereas species with neutral GC-content such as Salmonella enterica and Escherichia coli were only affected in certain regions. Species with high GC-content such as Mycobacterium tuberculosis and Pseudomonas aeruginosa were hardly affected at all. The coverage bias associated with Nextera XT was not found when Nextera Flex/DNA Prep had been used. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
3. The efficiency of Nextera XT tagmentation depends on G and C bases in the binding motif leading to uneven coverage in bacterial species with low and neutral GC-content
- Author
-
Bo Segerman, Ásgeir Ástvaldsson, Linda Mustafa, Joakim Skarin, and Hanna Skarin
- Subjects
Nextera XT ,uneven ,coverage ,GC ,bacterial ,genome ,Microbiology ,QR1-502 - Abstract
Whole-genome sequencing (WGS) is becoming the new standard for bacterial high-resolution typing and the performance of laboratories is being evaluated in interlaboratory comparisons. The use of the Illumina Nextera XT library preparation kit has been found to be associated with poorer performance due to a GC-content-dependent coverage bias. The bias is especially strong when sequencing low GC-content species. Here, we have made an in-depth analysis of the Nextera XT coverage bias problem using data from a proficiency test of the low GC-content species Campylobacter jejuni. We have compared Nextera XT with Nextera Flex/DNA Prep and examined the consequences on downstream WGS analysis when using different quantities of raw data. We have also analyzed how the coverage bias relates to differential usage of tagmentation cleavage sites. We found that the tagmentation site was characterized by a symmetrical motif with a central AT-rich region surrounded by Gs and Cs. The Gs and Cs appeared to be the main determinant for cleavage efficiency and the genomic regions that were associated with low coverage only contained low-efficiency cleavage sites. This explains why low GC-content genomes and regions are more subjected to coverage bias. We furthermore extended our analysis to other datasets representing other bacterial species. We visualized how the coverage bias was large in low GC-content species such as C. jejuni, C. coli, Staphylococcus aureus, and Listeria monocytogenes, whereas species with neutral GC-content such as Salmonella enterica and Escherichia coli were only affected in certain regions. Species with high GC-content such as Mycobacterium tuberculosis and Pseudomonas aeruginosa were hardly affected at all. The coverage bias associated with Nextera XT was not found when Nextera Flex/DNA Prep had been used.
- Published
- 2022
- Full Text
- View/download PDF
4. Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines.
- Author
-
Sukser, Viktorija, Rokić, Filip, Barbarić, Lucija, and Korolija, Marina
- Subjects
- *
MITOCHONDRIAL DNA , *HUMAN genome , *MOLECULAR genetics , *WORKFLOW , *FORENSIC genetics , *MOLECULAR diagnosis - Abstract
Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
5. GC content-associated sequencing bias caused by library preparation method may affect Salmonella serotype prediction using SeqSero2.
- Author
-
Shaoting Li, Shaokang Zhang, and Xiangyu Deng
- Subjects
- *
FORECASTING , *SALMONELLA , *SALMONELLA enterica , *SALMONELLA food poisoning , *EPITOPES , *MICROBIOLOGY - Published
- 2020
- Full Text
- View/download PDF
6. Developmental validation of a Nextera XT mitogenome Illumina MiSeq sequencing method for high-quality samples.
- Author
-
Peck, Michelle A., Sturk-Andreaggi, Kimberly, Thomas, Jacqueline T., Oliver, Robert S., Barritt-Ross, Suzanne, and Marshall, Charla
- Subjects
NUCLEOTIDE sequencing ,MITOCHONDRIAL DNA ,GENETIC markers ,QUALITY assurance ,NUCLEAR DNA - Abstract
Generating mitochondrial genome (mitogenome) data from reference samples in a rapid and efficient manner is critical to harnessing the greater power of discrimination of the entire mitochondrial DNA (mtDNA) marker. The method of long-range target enrichment, Nextera XT library preparation, and Illumina sequencing on the MiSeq is a well-established technique for generating mitogenome data from high-quality samples. To this end, a validation was conducted for this mitogenome method processing up to 24 samples simultaneously along with analysis in the CLC Genomics Workbench and utilizing the AQME (AFDIL-QIAGEN mtDNA Expert) tool to generate forensic profiles. This validation followed the Federal Bureau of Investigation’s Quality Assurance Standards (QAS) for forensic DNA testing laboratories and the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines. The evaluation of control DNA, non-probative samples, blank controls, mixtures, and nonhuman samples demonstrated the validity of this method. Specifically, the sensitivity was established at ≥25 pg of nuclear DNA input for accurate mitogenome profile generation. Unreproducible low-level variants were observed in samples with low amplicon yields. Further, variant quality was shown to be a useful metric for identifying sequencing error and crosstalk. Success of this method was demonstrated with a variety of reference sample substrates and extract types. These studies further demonstrate the advantages of using NGS techniques by highlighting the quantitative nature of heteroplasmy detection. The results presented herein from more than 175 samples processed in ten sequencing runs, show this mitogenome sequencing method and analysis strategy to be valid for the generation of reference data. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
7. Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines
- Author
-
Marina Korolija, Viktorija Sukser, Filip Rokić, and Lucija Barbarić
- Subjects
Mitochondrial DNA ,Guidelines as Topic ,Computational biology ,Biology ,DNA, Mitochondrial ,01 natural sciences ,Genome ,Workflow ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Humans ,030216 legal & forensic medicine ,mtDNA control region ,Massive parallel sequencing ,010401 analytical chemistry ,High-Throughput Nucleotide Sequencing ,Reproducibility of Results ,Sequence Analysis, DNA ,Molecular diagnostics ,0104 chemical sciences ,Minor allele frequency ,Genome, Mitochondrial ,Quality Score ,Human genome ,MiSeq ,mitochondrial DNA ,Nextera XT ,evaluation ,analysis thresholds - Abstract
Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics.
- Published
- 2021
8. A performance evaluation of Nextera XT and KAPA HyperPlus for rapid Illumina library preparation of long-range mitogenome amplicons.
- Author
-
Ring, Joseph D., Sturk-Andreaggi, Kimberly, Peck, Michelle A., and Marshall, Charla
- Subjects
NUCLEOTIDE sequencing ,MITOCHONDRIAL DNA ,FORENSIC sciences ,GENDER-nonconforming people ,DNA analysis - Abstract
Next-generation sequencing (NGS) facilitates the rapid and high-throughput generation of human mitochondrial genome (mitogenome) data to build population and reference databases for forensic comparisons. To this end, long-range amplification provides an effective method of target enrichment that is amenable to library preparation assays employing DNA fragmentation. This study compared the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA) for enzymatic fragmentation and indexing of ∼8500 bp mitogenome amplicons for Illumina sequencing. The Nextera XT libraries produced low-coverage regions that were consistent across all samples, while the HyperPlus libraries resulted in uniformly high coverage across the mitogenome, even with reduced-volume reaction conditions. The balanced coverage observed from KAPA HyperPlus libraries enables not only low-level variant calling across the mitogenome but also increased sample multiplexing for greater processing efficiency. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
9. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics
- Author
-
Christian Rinke, Serene Low, Ben J. Woodcroft, Jean-Baptiste Raina, Adam Skarshewski, Xuyen H. Le, Margaret K. Butler, Roman Stocker, Justin Seymour, Gene W. Tyson, and Philip Hugenholtz
- Subjects
Nextera XT ,100 fg ,Low input DNA library ,Picogram ,Reagent contamination ,Low biomass ,Medicine ,Biology (General) ,QH301-705.5 - Abstract
High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.
- Published
- 2016
- Full Text
- View/download PDF
10. Improvements in Next Generation Sequencing (7th Annual SFAF Meeting, 2012)
- Author
-
Fiske, Haley
- Published
- 2012
11. Generation of whole genome sequences of Knew Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples.
- Author
-
Hadfield, Stephen J., Pachebat, Justin A., Swain, Martin T., Robinson, Guy, Cameron, Simon J. S., Alexander, Jenna, Hegarty, Matthew J., Elwin, Kristin, and Chalmers, Rachel M.
- Subjects
- *
NUCLEOTIDE sequence , *CRYPTOSPORIDIUM , *OVUM , *IMMUNOMAGNETIC separation , *CRYPTOSPORIDIIDAE - Abstract
Background: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. Results: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. Conclusion: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
12. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics
- Author
-
Jean-Baptiste Raina, Ben J. Woodcroft, Xuyen H. Le, Justin R. Seymour, Gene W. Tyson, Margaret K. Butler, Serene Low, Adam Skarshewski, Philip Hugenholtz, Roman Stocker, and Christian Rinke
- Subjects
0301 basic medicine ,Library ,lcsh:Medicine ,Reagent contamination ,Computational biology ,Biology ,Microbiology ,General Biochemistry, Genetics and Molecular Biology ,Bottleneck ,Lower limit ,03 medical and health sciences ,chemistry.chemical_compound ,Illumina ,Environmental DNA ,Picogram ,Low volume ,Molecular Biology ,Microscale chemistry ,Low biomass ,Ecology ,General Neuroscience ,lcsh:R ,Low input DNA library ,General Medicine ,Genomics ,Nextera XT ,030104 developmental biology ,Community composition ,chemistry ,Metagenomics ,100 fg ,Microscale metagenomics ,General Agricultural and Biological Sciences ,DNA ,Marine microheterogeneity - Abstract
High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics., PeerJ, 4, ISSN:2167-8359
- Published
- 2016
13. GC Content-Associated Sequencing Bias Caused by Library Preparation Method May Infrequently Affect Salmonella Serotype Prediction Using SeqSero2.
- Author
-
Li S, Zhang S, and Deng X
- Subjects
- Base Composition, Gene Library, Serogroup, Serotyping, Salmonella genetics
- Published
- 2020
- Full Text
- View/download PDF
14. Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples
- Author
-
Simon J S Cameron, Matthew J. Hegarty, Rachel M. Chalmers, Martin T. Swain, Kristin Elwin, Guy Robinson, Justin A. Pachebat, Stephen J. Hadfield, and Jenna Alexander
- Subjects
Bioinformatics ,animal diseases ,Cryptosporidium ,Pilot Projects ,Genome ,DNA sequencing ,Microbiology ,Feces ,03 medical and health sciences ,Illumina ,parasitic diseases ,Genetics ,Humans ,030304 developmental biology ,Cryptosporidium parvum ,Whole genome sequencing ,0303 health sciences ,08 Information And Computing Sciences ,Base Sequence ,biology ,030306 microbiology ,Oocysts ,High-Throughput Nucleotide Sequencing ,11 Medical And Health Sciences ,DNA, Protozoan ,06 Biological Sciences ,Nextera XT ,biology.organism_classification ,DNA extraction ,Stool ,Immunomagnetic separation ,Cryptosporidium hominis ,Research Article ,Reference genome ,Biotechnology - Abstract
Background: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. Results: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. Conclusion: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.
- Published
- 2015
15. Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells.
- Author
-
Kim J and Daadi MM
- Subjects
- High-Throughput Nucleotide Sequencing, Transcriptome, Workflow, Gene Library, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Neural Stem Cells cytology, Neural Stem Cells metabolism, Single-Cell Analysis
- Abstract
Single-cell RNA-seq technology allows for the identification of heterogeneous cell populations, measures stochastic gene expressions, and identifies highly variable genes. Thus, with this technology it is possible to identify relevant pathways involved in development or in disease progression. Herein, we describe a protocol to capture and process single-cell transcriptomes that will be used for RNA sequencing. This chapter discusses the use of the Fluidigm C1 System and Integrated Fluidic Circuit microfluidics system, TapeStation 4200, SMART-Seq v4, Nextera XT Library Preparation Kit, and AMPure XP beads.
- Published
- 2019
- Full Text
- View/download PDF
16. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics.
- Author
-
Rinke C, Low S, Woodcroft BJ, Raina JB, Skarshewski A, Le XH, Butler MK, Stocker R, Seymour J, Tyson GW, and Hugenholtz P
- Abstract
High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics., Competing Interests: The authors declare that they have no competing interests.
- Published
- 2016
- Full Text
- View/download PDF
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