In the past few months we have recorded the nine protocol steps of the NGSgo Workflow. These videos visualize how to execute the workflow steps and can be used as an addition to the available Instructions for Use. The videos are now available on our website, please note that these videos are not a replacement for the Instructions for Use.

Remco Malestein B.ASc (Technical Application Specialist) and Jannetje Kooij B.ASc (Custom Laboratory Service Coordinator & Technician) have been using the NGSgo workflow for multiple years and are very happy to share some additional tips & tricks to optimize the results of your workflow.

Tip 1: Work in a dedicated DNA workplace
PCR amplification is a very sensitive technique, therefore you need to be aware of the possibility of sample contamination, which can have a negative impact on the results. In order to prevent contamination, we advise to separate the DNA from the amplification reagents in the pre-amplification lab. Move to a designated DNA workplace and wear a special DNA lab coat when you are going to dispense your DNA into the plate. Additionally, it is strongly recommended to perform pre-amplification and post-amplification procedures in separate rooms. In this way, the chance of contamination is minimized.

Tip 2: Standardization of DNA input
By standardizing the DNA input amount before amplification, the amplification results will be very consistent. For single plex amplification this makes the amplicon pooling very easy and consistent. For the multiplex amplifications MX6-1 and MX11-3 this will also yield very consistent sequencing results with similar read depths per sample.

Tip 3: Use 8-well strips
During the NGS workflow, when using a high amount of samples (>24), it will save time to first aliquot the ligase and/or HiFi mastermix in an 8-well strip. This way it is faster to distribute the mix in the plate using a multichannel pipet.

Tip 4: Beads at room temperature
At the start of the NGS workflow, don’t forget to take the beads that are needed in the next step (DNA cleanup) out of the refridgerator. The beads work optimal when at room temperature.

Tip 5: Use a multichannel with a reagent reservoir
If you want to save time during the first Bead Cleanup, make use of a reagent reservoir in combination with a multichannel pipet! This works well for pipetting the beads, elution buffer and 80% ethanol. By using the multichannel pipet you can  fill eight  tips at once which makes pipetting easy and faster. Any remaining beads or elution buffer can be re-used!

Tip 6: Use IndX plates
The Indexing PCR ensures that all samples have a unique barcode. When using the Library Full Kit, an IndX plate is included. This plate contains  all unique index primer combinations for a quick and easy setup of the Indexing PCR. This will save you a lot of time. Afterwards, simply use the GenDx Sample Sheet to link the sample name to used index combination.

Tip 7: Re-use the IndX plate up to 17 times
The IndX plate consists of 96 unique index combinations. A big advantage of this plate is that unused indices can be used at a later time. You can simply store the plate in the freezer and re-use it  up to 17 times! This means you can easily run a small amount of samples and save the leftover indices for later

Tip 8: Use Qubit quantification in the Illumina workflow
After your library preparation is finished the Quantification of your library can be done in two different ways. Using the KAPA qPCR assay, or the fluorescence based Qubit. Using the Qubit will save a significant amount of time. Simply using the correction factor to calculate the final concentration in nM (nanomolars)  and you are ready for sequencing!

Tip 9: Use Qubit correctly
The Qubit is an easy and fast fluorescence based quantification method. It is advised to use gloves when handling the Qubit tubes to prevent a dirty tube from influencing the measurement results. An extra tip is to put a glove over the vortex; in this way the Qubit tubes will not be smudged by the vortex rubber.

Tip 10: Use the flow cell calculation sheet
In order to most efficiently use the sequencer flow cell, GenDx has developed a flow cell calculation sheet. This excel tool will calculate the maximum amount of samples that can be loaded, no matter the loci configuration. The calculation is based on an minimum read depth of 500 reads.

After these 10 valuable tips & tricks, you are probably motivated to move to the lab and perform the NGSgo workflow! Use the Instruction for Use for the correct execution of the NGSgo Workflow and check out the Instructional videos here for a visualization of the steps.