Next Generation Sequencing High-throughput Solution for Illumina Fragment Library Sample Preparation

Scott Verrow, Kevin Boynton, Mary Blair, Kelly Marshall, Ruth Zhang, Alisa Jackson Beckman Coulter, Inc.

Automated Size Selection

Illumina library sample preparation for Next Generation Sequencing (NGS) is a long and complicated process that can alsorequire precise fragment size selection through agarose gel separations in order to obtain high quality sequencing results.

Beckman Coulter has released SPRIworks chemistry for low-throughput using the SPRI-TE Nucleic Acid Extractor and is now developing chemistry for high-throughput (HT) automation using Biomek® FXP liquid handlers for Illumina compatible NGS library sample preparation. The HT solution can produce up to 96 libraries in a 96-well format with minimum hands on time and uses Solid Phase Reversible Immobilization (SPRI) paramagnetic beads to offer various fragment sizes selection (150-350 bp, 250-450 bp, 350-700 bp, and 150 bp and up).Downstream processes, including PCR reaction setup and cleanup, qPCR setup, and normalization, have been automated to create a suite of Biomek methods. This suite of automation methods has asingle user interface (UI) to allow for easy selection of options.

Normalization and Pooling

PCR Clean-Up & qPCR Set-up

*All trademarks are property of the respective owners. **SPRIworks HT is in development. Biomek is for Laboratory use only; not for diagnostic procedures. SPRIworks is for Research Use Only. Beckman Coulter, the stylized logo, Biomek, SPRIworks and SPRI are trademarks of Beckman Coulter, Inc. and are registered in the USPTO.

SPRIworks HT Methods Suite

Conclusions

The SPRIworks HT Methods Suite offers the user the ability to run three different Biomek methods to prepare samples for loading onto the Illumina C-Bot. The first method covers the three steps of the sample preparation: library construction, size selection, and PCR setup without requiring any user intervention. The second method covers PCR Clean-Up using Ampure XP and qPCR setup using KapaBiosystems Library Quant Kit for Illumina. The third method utilizes the qPCR raw data to normalize the samples and pool any samples for multiplexing.

Figure 2: Example of the Library Construction (LC) User Interface Shows the following selected options:

All 3 modules selected, 96 Samples to process, variable size selection for each sample across the plate, 20 µL of sample to be used during PCR Setup

The UI provides a simple to use format for users to configure each SPRIworks HT run. Options such as the number of samples toprocess (1-96), Plate I.D., per sample size selection range, and sample volume for PCR setup are easily configurable.

Size selection ranges are written to a *.CSV file each time automated size select is performed. The *.CSV is imported into the downstream Normalization method. This enables users to create a more accurate quantitation for each sample by using chip data toenter an average base pair size for each size range selected.

Figure 1: Shows the initial UI screen. The three method options are listed to the side with a description box covering method details.

Library ConstructionThe starting input is clean fragmented gDNA supplied by the user.

The user may choose their preferred method of fragmentation and purification. (*Agencourt Ampure XP is the recommended purification process with validated Biomek methods available to use)

•The user loads the deck of the Biomek FXP with all the reagents necessary for LC. Each reaction occurs at room temperature.

•Ampure XP is used between each reaction to clean-up the samples for the next reaction.

The second method performs PCR clean-up using Ampure XP and prepares a 96-well qPCR plate using Kapa Biosystem’s KapaIllumina GA Library Quantification Kit. Users can select to runboth processes in one method run to save time.

Size Selection has been completely automated to allow for a walkaway solution that is reproducible. Four size selection ranges are available in the User Interface: No Selection, small, medium, and large. If electing to perform library construction and size selection together, selection will occur between each reaction. This format allows the user to save time and will allow selection to apply to the insert size. Downstream chip analysis of the samples will reflect the selected size range plus the size of adaptors combined.

Size selection can also be performed as a stand-alone method after library construction is completed. Selection will then be applied to samples already ligated to adaptors. Downstream chip analysis will reflect the size range selected

The tables below show results of analyzer chip data for each size range option. Samples were processed through library construction using integrated size selection and reflect the selected size range including the size of the adaptors.

Figure 4:

•PCR Clean-up can process up to 96 samples at once.

•qPCR Setup can process up to 24 samples in triplicate to insure quality quantification results.

The last method in the suite allows the user to normalize and pool samples. Users can elect to run both processes together or separately. The Normalization method uses the raw qPCR data to calculate quantity and to give the users the ability to make on-the-fly decisions regarding normalization. The Normalization method popsup an Excel* sheet that gives the quantities of each samples, and allows users to determine the final volume and concentration of the normalized plate. It also enables error handling to allow wells that do not meet the input volume or concentration to be skipped, or to continue normalization of low wells. Finally, it gives quick feedback on %CVs so outliers can be identified.

Figure 5: Example of Normalization Calculations Spreadsheet

•SPRIworks offers a complete package of chemistry and automated methods

Low-throughput and High-throughput automation solutions available

Chemistry and automation support from one company

•Automated size selection saves time and is reproducible

•User Interface allows for quick customization of various optionssuch as:

Number of samples processed up to 96 per run.

Size selection ranges assigned to each well

On-the-fly decisions for Normalization and Pooling.

Introduction

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gDNA Fragmented gDNA

Ampure XP Clean-up* Starting

Input End Repair

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Ampure XP Clean-up & A-Tailing

Ampure XP Clean-up & LigateAdaptors

Ampure XP Clean-up followed by Size Selection

Figure 3: Percent of Total is the area between the indicated range. Ranges reflect the insert size plus size of ligatedadaptors.