Downloads: App note and protocols for the effortless DNA library normalization using the ASSIST PLUS pipetting robot equipped with the D-ONE single channel pipetting module

Here you can download the complete protocols and reports used in this application and use them on your ASSIST PLUS and D-ONE. The protocol can be customized with the VIALAB software.

Overview: How to perform sample normalization for Illumina 16S metagenomic sequencing

This application note describes the automation of DNA library normalization. The protocol is based on Illumina's 16S Metagenomic Sequencing Library Preparation Guide. After quantifying the DNA libraries, the DNA concentrations should be calculated in nM concentration (see a calculation example for 500 bp long amplicons in the DNA concentration calculation Microsoft Excel® sheet). For sequencing, the libraries should be diluted to a concentration of 4 nM. The 'Normalization worklist' programming step allows the user to import a worklist with sample details and concentrations (Figure 1).

Step-by-step procedure:

The ASSIST PLUS pipetting robot is used together with the 0.5-300 µl D-ONE single channel pipetting module and 12.5 µl and 300 µl Sterile, Filter GRIPTIPS® to automate the pipetting steps of sample normalization for Illumina 16S metagenomic sequencing (Figure 3).

The normalization protocol consists of two steps:

1. Transfer of the diluent
2. Transfer of the samples

Experimental set-up

Deck position A: Diluent – 10 ml multichannel reagent reservoir

Deck position B: Source plate – Sapphire 96  well PCR plate (Greiner Bio-one)

Deck position C: Target plate – Sapphire 96  well PCR plate (Greiner Bio-one)

1.Transfer of diluent

The diluent is added in repeat dispense steps.

Place the 10 ml multichannel reagent reservoir on deck position A and fill it with 5 ml of 10 mM Tris (pH 8.5) buffer (Figure 4, blue with a white drop). Next, place the source plate (Sapphire 96 well PCR plate) with the amplicon samples on position B (Figure 4, colored circles) and an empty Sapphire 96 well PCR plate on position C (Figure 4, colored circles with dots); this will be the target plate containing the diluted, normalized samples.

Select and run the VIALAB program 'Illumina amplicon normalization'. The system first transfers the calculated diluent volumes to each well in several repeat-dispense steps with tip touch (Figure 5). A 15  µl pre- and post-dispense ensures precision while pipetting, and the total volume for each aspiration is calculated automatically. This is the fastest way of dispensing different volumes with a single channel pipette, saving time and money while reducing tip usage.

Tip:

• Pre- and post-dispense settings, together with the tip touch, guarantee precise and accurate pipetting results.

2. Transfer of the samples

The samples are transferred one by one.

After dispensing the diluent, the ASSIST PLUS pipetting robot starts to add the amplicon samples from the source plate to the target plate in individual transfer steps (Figure 6).

The amplicon sample volumes in the source plate may be different. If the liquid level detection (LLD) (Figure 7) is activated, the optimal pipetting height is automatically detected and allows the pipette tip to follow the liquid level during aspiration. This constant submerge depth during aspiration contributes to precise pipetting results. At the end of every transfer step, the sample is mixed thoroughly with the diluent in three mixing cycles.

Tip:

• Pipetting parameters (pipetting speed, pre-wetting, tip change) and the mixing conditions (height, speed, cycle and volume) can easily be adjusted in the VIALAB program if needed.

Now the normalized libraries are ready to be aliquoted and pooled for sequencing.

Remarks

• VIALAB software: VIALAB programs can be easily adapted to the user’s specific labware and protocols.
• Run report: If the ASSIST PLUS pipetting robot is connected to the PC with VIALAB, programs can be started directly from the PC. After the run, a run report is automatically generated, documenting details such as the start/end time, user, calculated volumes, and any errors that occurred. This offers a convenient way to fulfill regulatory requirements.