Biomek i-Series Automated Illumina TruSeq® Nano DNA Library Prep Kit
Illumina TruSeq Nano DNA Library Prep Kit enables preparation of uniquely indexed DNA libraries for single end and paired end sequencing (Figure 1). Especially designed for low sample input, this method is known to deliver high coverage across the genomes and reduce library bias (Illumina TruSeq® Nano DNA Library Prep Guide15041110-d). The method avoids the sample loss associated with gel-based selection by the use of a bead-based size selection strategy. Therefore, the Nano DNA Library Prep Kit is widely used across many NGS applications. In this technical note, we will demonstrate automated performance of the Kit on the Biomek i7 Dual Hybrid (Multichannel 96, Span-8) Genomics Workstation.
When compared to manual pipetting, the Illumina TruSeq Nano DNA Library Prep Kit automated on a Biomek platform provides:
Figure 1. Illumina TruSeq® Nano DNA Library Prep Kit protocol.
|24 Samples||96 Samples|
|Prepare Reagents, Set up Instrument||15 min||30 min|
|Method Run (walk away time)||4 hr, 52 min||5 hr, 51 min|
|Total*||5 hr, 7 min||6 hr, 21 min|
*Timing estimate includes incubations and thermocycling.
Timing estimate does not include reagent thawing.
Table 1. Estimated run times for Illumina TruSeq® Nano DNA Library Prep Kit on the Biomek i7 Dual Hybrid Genomics Workstation.
Figure 2. Biomek Method Launcher provides an easy interface to start the method
Figure 3. Biomek Method Options Selector indicate sample number and processing options
Automation provides increased efficiency, reducing the hands-on time (Table 1). The automated TruSeq Nano DNA Library Prep Kit protocol includes logical start and stop points assigned based on Illumina’s recommendations, providing the users flexibility in scheduling their day (Figure 1).
1. Biomek Method Launcher (BML)
BML is a secure interface for method implementation without introducing errors during method setup (Figure 2). Within BML, the method steps are organized in a modular manner for workflow optimization (Figure 1).
2. Method Options Selector (MOS)
MOS enables selection of sample number and sample processing options (e.g. insert size selection, adaptor transfer options) to maximize flexibility, adaptability and the ease of method execution (Figure 3). The thermocycling steps can be done either off-deck or on-deck using an automated thermocycler (ATC Thermo Fisher) (Figure 4).
Figure 4. Deck Layout for Illumina TruSeq® Nano DNA Library Prep Kit protocol on Biomek i7 Dual Hybrid for 96 samples with on-deck thermocycling option
3. Guided Labware Setup (GLS)
GLS provides the user specific text and graphical setup instructions with reagent volume calculation and step by step instructions to prepare reagents (Figure 4). The automated method supports placing Illumina low throughput and high throughput adaptor labware on deck along with custom adaptor plates (Figure 5). The GLS steps are generated based on the options selected in MOS. For instance, selecting automatic adaptor transfer creates dataset driven adaptor ID logs. Users also have the ability to customize adaptor assignments by uploading a .csv file.
Figure 5. Guided Labware Setup indicates calculated reagent volumes required to make reagent mixes and guides the user for correct deck setup
Figure 6. Guided Labware Setup enables selecting index tube layout
Promega Human gDNA (200 ng/µl) was used for the automation of the Nano DNA Library Prep kit protocol on the Biomek i7 Dual Hybrid (Multichannel 96, Span-8) Genomics Workstation. After the preparation, the libraries were analyzed on Agilent TapeStation 2200 with High Sensitivity D5000 Kit and by qPCR (Applied Biosystems 7900HT Fast real time PCR system) using KAPA Library Quantification Kit.
Agilent TapeStation results indicated that the prepared libraries are of expected size (Approximately around 900 base pairs, Illumina TruSeq® Nano DNA Library Prep Guide15041110-d; Table 2; Figure 7). The yields according to qPCR show that the libraries are suitable for sequencing (Approximately 10nM of samples are used for the proceeding steps, Illumina TruSeq® Nano DNA Library Prep Guide15041110-d; Table 2).
|Sample ID||Mass (ng)||Insert Size (bp)||Index||TapeStation Size (bp)||qPCR Yield (nM)|
|Promega Human gDNA rep1||200||550||AD001||845||452|
|Promega Human gDNA rep2||200||550||AD003||917||383.4|
|Promega Human gDNA rep3||200||550||AD008||960||383.4|
|Promega Human gDNA rep4||200||550||AD009||547||365.3|
|Promega Human gDNA rep5||200||550||AD010||958||549.8|
|Promega Human gDNA rep6||200||550||AD011||849||398.8|
|Promega Human gDNA rep7||200||550||AD020||879||389.8|
|Promega Human gDNA rep8||200||550||AD022||842||453.5|
|Promega Human gDNA rep9||200||550||AD023||838||476.6|
|Promega Human gDNA rep10||200||550||AD025||846||481.3|
|Promega Human gDNA rep11||200||550||AD027||837||454.5|
|Promega Human gDNA rep12||200||550||AD002||812||401.6|
|Promega Human gDNA rep13||200||550||AD004||852||404.9|
|Promega Human gDNA rep14||200||550||AD005||905||393.9|
|No Template Control||0||550||AD006||0||0|
Table 2. Library quantification of automated Illumina TruSeq® Nano DNA Library Prep kit protocol using Agilent TapeStation 2200 and qPCR using the Kapa Illumina Library Quantification Kit.
Figure 7. Electropherogram (Sample intensity vs. size in base pairs) of Agilent TapeStation corresponding to UHR250 replicate3 showing the libraries around expected size of the marker