Integrated magneto–electrophoresis microfluidic chip purification on library preparation device for preimplantation genetic testing for aneuploidy detection

Schneider, Lindsay; Fraser, Michelle; Tripathi, Anubhav

doi:10.1039/d1ra01732b

Cited by 4 publications

(5 citation statements)

References 38 publications

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“…NGS is a markedly valuable tool for diagnostics and discovery because it generates millions of reads and is exceedingly sensitive to nucleic acid mutations [9][10][11][12] . Unfortunately, while innovations in sequencing technology have reduced requisite cost, time, and workload, sample (library) preparation has largely remained rudimentary and labor-intensive [13][14][15][16] . Even for applications requiring less data, such as microbial whole genome sequencing (WGS) or RNA-seq, the majority of the total sequencing cost can be attributed to library preparation,…”

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confidence: 99%

“…1E). A post-PCR purification step is necessary to remove unwanted primer and adapter dimers, nucleotides, and any DNA fragments less than 200 bp in length 14,16 .…”

mentioning

confidence: 99%

“…Small laboratories are consequently often restricted to labor-intensive manual protocols that introduce many opportunities for sample contimination and manual pipetting errors due to various factors. Microfluidic approaches are one group of solutions often more suitable for low-throughput and single-use library preparation applications 14,16,24,[27][28][29] . Single-use microfluidic chips harbor risks for the environment, producing consequential waste and requiring safe disposal if toxic or dangerous chemicals are incorporated into chip design.…”

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confidence: 99%

“…Consequently, expensive robotic liquid handlers dominate research and clinical applications in core laboratories, and small laboratories suffer from the bottleneck of labor-intensive manual protocols. Automation innovation for small laboratories is necessary to accelerate NGS-based research, eliminate sources of human error, and reduce hands-on time [13][14][15][16] . Our previous work started with the innovative concept of integrating liquid mixing steps with thermodynamically driven steps such as ligation and amplification via a single capillary tube heating system [13][14][15][16] .…”

mentioning

confidence: 99%

“…Automation innovation for small laboratories is necessary to accelerate NGS-based research, eliminate sources of human error, and reduce hands-on time [13][14][15][16] . Our previous work started with the innovative concept of integrating liquid mixing steps with thermodynamically driven steps such as ligation and amplification via a single capillary tube heating system [13][14][15][16] . This work utilized magnetic bead motion through a microchannel for purification step, as needed by steps C and D in Fig.…”

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confidence: 99%

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Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation

Ott,

Tolppi,

Figueroa-Cruz

et al. 2024

Sci Rep

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Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption–desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations.

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confidence: 99%

“…1E). A post-PCR purification step is necessary to remove unwanted primer and adapter dimers, nucleotides, and any DNA fragments less than 200 bp in length 14,16 .…”

mentioning

confidence: 99%