Automation of Amplicon-Based Library Preparation for Next-Generation Sequencing by Centrifugal Microfluidics

Abstract

Next-generation sequencing (NGS) has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottleneck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic cartridge. Multiplex polymerase chain reaction amplification and subsequent cleanup were performed with all reagents prestored on the disk, including cell-line-based DNA as quality control. Exchange of prestored reagents allows applying the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent cleanup performance between 91.9 and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward primers combined with 4 reverse primers. The fully automated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.

Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology

“…This technology provides the advantages of minimizing the DNA and reagent requirement to nanoliters along with automation and consolidation of the PCR reaction onto one nanofluidic chip. Post-PCR, the amplicons from each sample can be retrieved from the reaction compartments and processed further for sequencing [ 21 , 22 ]. Another specialized PCR technology that has been adapted for NGS target enrichment is anchored multiplex PCR, where the amplification is open-ended: only one side of the ROI sequence is targeted using a target-specific primer (anchor), while the other end is targeted with a universal primer.…”

Analysis of the gut microbiome in dogs and cats

“…Because conserved regions are targeted, in theory, DNA from known and unknown bacteria present in the sample can be amplified, and then variable regions can be sequenced. This process requires specific library preparations for different sequencing platforms (eg, Illumina MiSeq, Ion Torrent PGM) 65 . The obtained raw sequences are then processed through a bioinformatics pipeline, such as QIIME 2 or Mothur, 66,67 to eliminate sequences of insufficient quality and erroneous reads, remove chimeric sequences, and compare the final sequences against public databases 60,68,69 .…”

“…This process requires specific library preparations for different sequencing platforms (eg, Illumina MiSeq, Ion Torrent PGM). 65 The obtained raw sequences are then processed through a bioinformatics pipeline, such as QIIME 2 or Mothur, 66 , 67 to eliminate sequences of insufficient quality and erroneous reads, remove chimeric sequences, and compare the final sequences against public databases. 60 , 68 , 69 Statistical analysis is performed according to the study design.…”

Advantages and Limitations of 16S rRNA Next-Generation Sequencing for Pathogen Identification in the Diagnostic Microbiology Laboratory: Perspectives from a Middle-Income Country

“…In addition, automation in liquid handling for library preparation is achievable via pipetting workstations such as the Biomek i-Series (Beckman Coulter and Bravo Automated Liquid Handling Platform (Agilent) [53]. In future, microfluidics solutions for NGS library preparation will enable miniaturization and enclosed environment for the process, minimizing contamination and optimizing laboratory space utilization [54][55][56].…”