Preparation of Sequencing Library Pool

The application below will help with the pooling of RNA BaseCode libraries. It assumes that you have performed both PCR A and PCR B for each sample and have measured concentrations and fragment lengths of the resulting libraries.

To prepare the sequencing library pool for Illumina sequencing, the calculator will help you combine the purified PCR A and PCR B reactions from each sample to a sequencing library pool.

For MGI DNBSEQ, PCR A and PCR B libraries are separately pooled and circularized. The resulting circular ssDNA PCR A and PCR B pools are finally pooled to produce the sequencing library pool that can be used for DNB making and sequencing using the MGI DNBSEQ platform.

How do I use the calculator?

  1. Before you start pooling, check that the indices you have selected are compatible and that there are no index collisions between samples.

  2. The default molarity, volume and femtomole targets are typical, but your sequencing service provider may have other expectations, so please confirm this before the library pooling.

  3. Set the number of samples for which you have purified PCR A and a PCR B reactions.

  4. Fill in the concentrations and fragment length information.

  5. Confirm that the resulting femtomole yield of the pooling is sufficient for your sequencing service provider. If it is not, increase volume or molarity as needed.

  6. Pipette the appropriate volume of water for each pool into 1.5mL Eppendorf Tubes.

  7. Pipette the appropriate volume of each library to obtain the sequencing library pool.

  8. In case of MGI DNBSEQ sequencing:

    • Circularize pool A and Pool B separately

    • Enter resulting ssDNA concentration

    • Pipette the appropriate amounts of Pool A, Pool B and water to obtain the sequencing library pool.

Diagram illustrating the two approaches to DNA sequencing, Illumina and MGI DNB-seq. The Illumina process involves PCR amplification of DNA to create a sequencing library pool, which is then sequenced. The MGI DNB-seq approach uses PCR to create separate pools, then performs linear and circular DNA libraries before pooling for sequencing.

User Guide for calculator