LD-Based Genotype and Haplotype Inference from Low-Coverage Short Sequencing Reads
Bacterial Whole-Genome Sequencing Infographic · • De novo assembly from short-read sequencing...
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Application: Bacterial Whole-Genome Sequencing Data on file. *Automation and Connectivity solutions are in development. KAPA and LightCycler are For Research Use Only. Not for use in diagnostic procedures. Solutions KAPA HyperPlus Kits with KAPA HiFi HotStart ReadyMix offer rapid, flexible and low-bias library construction, from extracted DNA or crude bacterial lysates. KAPA Dual-Indexed Adapters are QC-tested for NGS performance and barcode cross-contamination. KAPA Pure Beads offer efficient, tunable reaction cleanups. KAPA Library Quantification Kits enable accurate, qPCR-based library quantification. Compatible with Roche LightCycler ® 96 and LightCycler ® 480 real-time PCR systems. Application Challenges • Bacteria have very diverse cellular and genomic architecture. Robust methods are needed to process a wide range of species and strains in standardized sequencing pipelines. • Liquid culture of bacterial isolates and DNA extraction for WGS library preparation are time consuming and may add cost to the workflow. • Many important pathogens have AT- or GC-rich genomes. Biases introduced during sample preparation and sequencing makes it difficult to obtain reliable coverage of certain genomic regions or genes of interest. • De novo assembly from short-read sequencing technologies is inherently difficult, and more so when contigs are short and large stretches of genomic content are missing from sequence data. • Bacterial WGS is often peformed in high-throughput settings that require flexibility and quick turnaround times. Benefits • Rapidly prepare high-quality WGS libraries from extracted DNA, or directly from crude bacterial lysates to significantly reduce the turnaround time from bacterial isolate to sequence. • Eliminate the need for mechanical fragmentation with the KAPA HyperPlus Kit with integrated enzymatic fragmentation. Increase thoughput with a flexible, automation-ready protocol, that can be tailored to produce insert sizes that are optimal for your sequencing read length. • Introduce minimal bias during fragmentation and library amplification with KAPA Frag and KAPA HiFi enzymes. Higher uniformity typically translates to better coverage of difficult genomic regions, and longer contigs for de novo assembly. • Enjoy greater peace of mind with integrated support and service for a complete bacterial WGS workflow solution. Sample Sequencing Ready Library Automation & Connectivity S a m p l e C o ll e c t i o n S a m p l e E n r i c h m e n t N u c l e i c A c i d E x t r a c t i o n S a m p l e Q u a n t i fi c a t i o n / Q C L i b r a r y P r e p a r a t i o n T a r g e t E n r i c h m e n t L i b r a r y Q u a n t i fi c a t i o n * GC-bias plot for Clostridium difficile (29% GC) whole-genome libraries, prepared with the KAPA HyperPlus Kit (green), or kits from other suppliers (red and orange). With the Roche Sample Prep Solution, very even coverage was achieved across all regions of the AT-rich genome. Libraries prepared with other sample prep methods are missing more genomic content regions, and must be subjected to additional sequencing to produce the same average coverage. Normalized Coverage GC% of 100 Window Bins 40 45 50 10 35 30 25 20 15 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Number of Windows (x 10 3 ) 300 250 200 150 100 50 0 Roche Sample Prep Workflow
Transcript of Bacterial Whole-Genome Sequencing Infographic · • De novo assembly from short-read sequencing...
Application:Bacterial Whole-Genome Sequencing
Benefits of Roche Workflow Solution:
Data on file.*Automation and Connectivity solutions are in development.KAPA and LightCycler are For Research Use Only. Not for use in diagnostic procedures.
Solutions
KAPA HyperPlus Kits with KAPA HiFi HotStart ReadyMix offer rapid, flexible and low-bias library construction, from extracted DNA or crude bacterial lysates.
KAPA Dual-Indexed Adapters are QC-tested for NGS performance and barcode cross-contamination.
KAPA Pure Beads offer efficient, tunable reaction cleanups.
KAPA Library Quantification Kits enable accurate, qPCR-based library quantification. Compatible with Roche LightCycler® 96 and LightCycler® 480 real-time PCR systems.
Application Challenges
• Bacteria have very diverse cellular and genomic architecture. Robust methods are needed to process a wide range of species and strains in standardized sequencing pipelines.
• Liquid culture of bacterial isolates and DNA extraction for WGS library preparation are time consuming and may add cost to the workflow.
• Many important pathogens have AT- or GC-rich genomes. Biases introduced during sample preparation and sequencing makes it difficult to obtain reliable coverage of certain genomic regions or genes of interest.
• De novo assembly from short-read sequencing technologies is inherently difficult, and more so when contigs are short and large stretches of genomic content are missing from sequence data.
• Bacterial WGS is often peformed in high-throughput settings that require flexibility and quick turnaround times.
Benefits
• Rapidly prepare high-quality WGS libraries from extracted DNA, or directly from crude bacterial lysates to significantly reduce the turnaround time from bacterial isolate to sequence.
• Eliminate the need for mechanical fragmentation with the KAPA HyperPlus Kit with integrated enzymatic fragmentation. Increase thoughput with a flexible, automation-ready protocol, that can be tailored to produce insert sizes that are optimal for your sequencing read length.
• Introduce minimal bias during fragmentation and library amplification with KAPA Frag and KAPA HiFi enzymes. Higher uniformity typically translates to better coverage of difficult genomic regions, and longer contigs for de novo assembly.
• Enjoy greater peace of mind with integrated support and service for a complete bacterial WGS workflow solution.
Sample
Sequencing Ready Library
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tificat
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et En
richment
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ntification
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GC-bias plot for Clostridium difficile (29% GC) whole-genome libraries, prepared with the KAPA HyperPlus Kit (green), or kits from other suppliers (red and orange). With the Roche Sample Prep Solution, very even coverage was achieved across all regions of the AT-rich genome. Libraries prepared with other sample prep methods are missing more genomic content regions, and must be subjected to additional sequencing to produce the same average coverage.
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GC% of 100 Window Bins40 45 5010 3530252015
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03 )
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e Pr
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Application Bacterial Whole-Genome Sequencing
Unlock the Potential of Every Sample
Application: Bacterial Whole-Genome Sequencing
Unlock the Potential of Every Sample
Published by:
Roche Sequencing Solutions, Inc. 4300 Hacienda Drive Pleasanton, CA 94588
sequencing.roche.com
Ordering information
Roche Cat. No. Kapa Code Description Pack Size
07962401001 KK8512 KAPA HyperPlus Kit 24 reactions
07962410001 KK8513 KAPA HyperPlus Kit, PCR-free 24 reactions
07962428001 KK8514 KAPA HyperPlus Kit 96 reactions
07962436001 KK8515 KAPA HyperPlus Kit, PCR-free 96 reactions
08278555702 KK8722 KAPA Dual-Indexed Adapter Kit (15 μM) 96 x 20 μL
07983280001 KK8001 KAPA Pure Beads 30 mL
07983298001 KK8002 KAPA Pure Beads 60 mL
07960140001 KK4824 KAPA Library Quantification Kit (Illumina®/Universal) 500 reactions
07960298001 KK4854 KAPA Library Quantification Kit (Illumina/LC480) 500 reactions
Unlock the Potential of Every SampleAs the first step in the NGS workflow continuum, sample prep holds the key to unlocking the potential of every sample. Because NGS samples are precious, the best methods are needed to process more samples successfully, obtain more information from every sample, and optimize your sequencing resources. From sample collection to sequencing-ready libraries, Roche Sample Prep Solutions offer workflows for different sample types and sequencing applications that are proven, simple and complete.
PublicationsJones, M.B. et al. Library preparation methodology can influence genomic and functional predictions in human microbiome research. Proc. Natl. Acad. Sci. USA. 2015;112:14024. doi:10.1073/pnas.1519288112.Grevskott, D.H. et al. Marine Bivalve Mollusks As Possible Indicators of Multidrug-Resistant Escherichia coli and Other Species of the Enterobacteriaceae Family. Front. Microbiol. 2017;8:24. doi:10.3389/fmicb.2017.00024.Bårnes, G.K. et al. Whole genome sequencing reveals within host genetic changes in paired meningococcal carriage isolates from Ethiopia. BMC Genomics. 2017;18:407. doi:10.1186/s12864-017-3806-3.
App NotesA novel, single-tube enzymatic fragmentation and library construction method enables fast turnaround times and improved data quality for microbial whole-genome sequencing. Roche Sequencing Solutions, 2016.The KAPA HyperPlus Kit enables a streamlined bacterial whole-genome sequencing workflow, utilizing crude lysates from single colonies. Roche Sequencing Solutions, 2017.
WebinarThe rise of whole-genome microbial analysis: A new era for human microbiome analysis, featuring Dr. Julie Segre (Microbial Genomics, US National Institutes of Health National Human Genome Research Institute) and Dr. Katie Pollard (Gladstone Institutes and University of California in San Francisco). Presented by Science and Roche Sequencing Solutions, 4 October 2017.
Data on file.KAPA and LightCycler products are For Research Use Only. Not for use in diagnostic procedures.KAPA and LIGHTCYCLER are trademarks of Roche.All other product names and trademarks are the property of their respective owners.©2018 Roche Sequencing Solutions, Inc. All rights reserved. SEQ100264 05/18
