Life Science


by Sikander Gill, Rajwant Gill, Nick Bandy and Dong Liang


Validation of a Magnetic Bead Mixer on an Automated Next-Generation Sequencing Library Preparation System


R


educed costs and higher throughput have made next- generation sequencing (NGS) more accessible to users in such diverse fields as diagnostics, infectious disease, food safety


and public health. NGS has revolutionized these areas as a result of its ability to produce genomes on an unprecedented scale1-4


in less time


and at a lower cost. Increased capacity has created a challenge: es- tablishment of an automated library preparation workflow to enable reliable and robust sequencing.


For every application of NGS there is a specific protocol to convert the source nucleic acid to a standard DNA library. The basic NGS library preparation pro- cedure involves nucleic acid isolation, adapter and bar-code ligation, DNA fragment size selection, amplification and amplification reaction cleanup.


Magnetic beads play a significant role in many of these steps, highlighting the need for their uniform distribution (Table 1). An efficient bead mixer placed on the deck of a robotic workstation can potentially keep the beads uniformly suspended in the source tube or reservoir before being distributed to the target wells of the library preparation plate.5


Since the


beads are precious and supplied in small volume, the mixer is expected to minimize dead volume. In addition, bead mixers provide advantages over pipet mixing, as the latter results in the beads clumping or sticking inside the pipet tips, which leads to inefficient mixing and nonuniform distribution of the beads onto the target plate.


The study presented in this article demonstrates use of a bead mixer installed on the VERSA 1100 NGS library preparation workstation (Aurora Biomed, Vancouver, B.C.).


Materials and methods


Determination of uniform bead distribution The materials and methods from Gill et al.5


were used to validate a bead


mixer installed on the VERSA 1100 workstation. The magnetic beads were suspended in 5 mL of lysis solution used for nucleic acid isolation; the tube containing the beads was inserted into the bead mixer. Next, using the system’s single-channel functionality, 20 µL of beads was automatically distributed from the bead mixer to each well of a 96-well multiple-well plate. The sampling was carried out in replicates of n = 10 to check the uniform distribution of the beads. The beads from each well of the target plate were resuspended in 1 mL of distilled water to read OD600 Spectrumlab 22PC spectrophotometer (Spectrumlab, Shanghai, China).


on the


Automated NGS library preparation The VERSA 1100 NGS automated liquid handling library preparation workstation was further evaluated for its ability to produce high-quality,


Table 1 – Applications of magnetic beads in next-generation sequencing


Unraveling the genomic targets of small molecules6 High-resolution digital profiling of the epigenome7


Sequencing pools of individuals—mining genome-wide polymorphism8


Isolation of mutant genes from forward genetic screens9 Genomic DNA sequencing of microbial species from single cells10


Whole-genome sequencing on the reconstruction of human population history11


Comparative primate genomics12


Ribosome profiling: new views of translation, from single codons to genome scale13


Bacterial genome sequencing in the clinic14 Single-cell sequencing of whole-organism science15 Cancer genome-sequencing study design16 Disease-targeted sequencing17


De novo mutations in human genetic disease18


Sequencing of human microbiome for at the interface of health and disease19 Protein–RNA interactions20 Exome sequencing21 RNA sequencing22


Charting histone modifications and the functional organization of mammalian genome23


Prokaryotic transcriptomics24


consistent libraries for NGS on the Ion Torrent PGM (Life Technologies, Grand Island, N.Y.). In the NGS library preparation protocol, the bead mixer was used for cleanup procedures for both the unamplified and amplified libraries, including first-round purification, second-round purification and fragment size selection as per the manufacturer’s protocol. Agencourt AMPure XP magnetic beads (Beckman Coulter Inc., Brea, Calif.) were used for cleanup. The Ion AmpliSeq library preparation kit and Ion AmpliSeq ready‐to‐use cancer panel kit (Life Technologies Inc.) screened the loci listed for allelic frequency associated with cancer hot spots. The Ion PGM sequencer (Life Technologies) was used to generate the NGS data.


AMERICAN LABORATORY • 6 • MARCH 2015


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56