scholarly journals A Microfluidics Workflow for Sample Preparation for Next-Generation DNA Sequencing

2018 ◽  
Vol 24 (2) ◽  
pp. 196-208 ◽  
Author(s):  
Adam Snider ◽  
Michael Nilsson ◽  
Mark Dupal ◽  
Masoud Toloue ◽  
Anubhav Tripathi
Keyword(s):  
Sample Preparation ◽  
Magnetic Bead ◽  
Library Preparation ◽  
Next Generation ◽  
Preparation Methods ◽  
Next Generation Sequencing Technology ◽  
Extraction And Purification ◽  
Spin Column ◽  
Sequencing Library ◽  
End Sequences

Next-generation sequencing technology requires amplified, short DNA fragments with known end sequences. Samples must undergo processing steps, including extraction and purification of genomic DNA (gDNA), fragmentation, end repair, adapter ligation, and amplification, to prepare a sequencing library. The process of sample preparation requires careful control of temperature and buffer conditions, as well as the stringent removal of contaminants. As a result, library preparation methods are often plagued by sample loss, long protocol times, numerous manual steps, and high cost. We attempt to understand and optimize each step of sample preparation on a microfluidic platform using magnetic bead motion through channels containing immiscible phases. Our platform integrates all steps associated with library preparation with no buffer exchanges and utilizes just 30–60 µL of reagents. Our chip shows a sixfold improvement in yield compared with an affinity spin column when capturing gDNA from samples of ~50 ± 4 MCF-7 cells. Finally, we show whole-genome shotgun sequencing results from 660 pg of human gDNA, in which >93 ± 1% of reads map to a reference genome at or above 99.9% confidence, matching a commercially available sample preparation kit optimized for low-cell-count samples.

Next Generation Sequencing Technology in Clinical Diagnostics

2020 ◽  
Vol 2 (7) ◽  
pp. 10-17
Author(s):  
Megha Agrawal ◽  
Keyword(s):  
Next Generation Sequencing ◽  
Basic Research ◽  
Cost Effective ◽  
Clinical Diagnostics ◽  
Library Preparation ◽  
Next Generation ◽  
Sequencing Technology ◽  
Next Generation Sequencing Technology ◽  
Diagnostic Applications ◽  
Generation Sequencing

The promise of next generation sequencing offers hope for the current DNA sequencing technology to evolve from the applications in basic research to transition to the clinical diagnostics. This advancement in the sequencing technology is happening in part due to the introduction of high throughput and benchtop instruments that offer fully automated cost-effective sequencing along with faster assay times. This development is believed to remove the bottleneck of the complex and cumbersome library preparation that include isolation of nucleic acids and the resulting amplified and barcoded DNA with sequencing adapters. Here, we present a brief overview of the principles of next generation sequencing and automation of library preparation along with the diagnostic applications of next generation sequencing in human immunogenetics. Finally, an outlook is presented.

scholarly journals qPCR-based characterization of DNA fragmentation efficiency of Tn5 transposomes

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Vera Rykalina ◽  
Alexey Shadrin ◽  
Hans Lehrach ◽  
Tatiana Borodina
Keyword(s):  
Next Generation Sequencing ◽  
Dna Fragmentation ◽  
Fragment Size ◽  
Library Preparation ◽  
Next Generation ◽  
Amplification Curve ◽  
Fragmentation Efficiency ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing

Abstract Here, we describe an electrophoresis free assay for characterizing Tn5 transposomes fragmentation efficiency in a tagmentation reaction, in which double-stranded DNA is fragmented and tagged with adapter sequences. The assay uses plasmid DNA as a reference tagmentation substrate. Fragmentation efficiency is analyzed by comparative qPCR which measures the difference (ΔCt) in amplification of a specific plasmid region before and after tagmentation: more efficient fragmentation is characterized by a larger number of cleavage events within the amplified region, a delayed increase in the amplification curve and as a result, a larger ΔCt. Tagmentation reactions characterized with the same ΔCt exhibit the same fragment size distribution on an agarose gel. The ΔCt values measured can be used to quantitatively determine the relative performance of Tn5 transposome assemblies in optimization experiments and to standardize between batch variations in transposomes for use in next-generation sequencing library preparation. Moreover, the use of a reference tagmentation template added during next-generation sequencing library preparation enabled monitoring of the input DNA fragmentation. The presented qPCR-based assay is quick, contamination-safe, high-throughput and cost-efficient.

scholarly journals Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes

BMC Genomics ◽  
2012 ◽  
Vol 13 (1) ◽  
pp. 1 ◽  
Author(s):  
Samuel O Oyola ◽  
Thomas D Otto ◽  
Yong Gu ◽  
Gareth Maslen ◽  
Magnus Manske ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Library Preparation ◽  
Next Generation ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing

scholarly journals Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

2020 ◽  
Author(s):  
Elizabeth M. Batty ◽  
Theerarat Kochakarn ◽  
Arporn Wangwiwatsin ◽  
Khajohn Joonlasak ◽  
Angkana T. Huang ◽  
...  
Keyword(s):  
Variant Calling ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Library Preparation ◽  
Preparation Methods ◽  
Base Calling ◽  
Sequencing Library ◽  
Miseq Platform ◽  
Downstream Analysis ◽  
Sequencing Library Preparation

AbstractGenomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

scholarly journals Comparison of Sample Preparation Methods Used for the Next-Generation Sequencing of Mycobacterium tuberculosis

PLoS ONE ◽  
2016 ◽  
Vol 11 (2) ◽  
pp. e0148676 ◽  
Author(s):  
Andrea D. Tyler ◽  
Sara Christianson ◽  
Natalie C. Knox ◽  
Philip Mabon ◽  
Joyce Wolfe ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Next Generation Sequencing ◽  
Sample Preparation ◽  
Next Generation ◽  
Preparation Methods ◽  
Generation Sequencing ◽  
Sample Preparation Methods
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