Using NGS for CE sequencing

Eureka Genomics’ highly multiplexed sample indices allow you to use next generation sequencing for CE sequencing. The inexpensive per base cost of next generation sequencing make this an economical alternative to CE sequencing. The indices can be broadly applied to sequencing needs. Currently there are 2,304 indices compatible with Illumina NGS platforms.

If 2,304 samples were put on a single MiSeq run, this would be an average of 10,000+ reads per sample. Clearly, more than is needed for most applications. The Eureka Genomics sample indices are compatible with the 48 RPI Illumina index barcodes, including the 12 TruSeq indices. Eureka Genomics sample indices are also compatible with other barcode sets such as Illumina D5xx-D7xx and the Nugen Encore 384 barcode set. Amplicons with these indices can be put in a lane/ run other libraries, as long as the other libraries are multiplexed using standard Illumina barcodes and the index read (typically R2 on GAIIx/HiSeq/MiSeq platforms) is 12 cycles.

The Eureka Genomics sample indices do NOT need special sequence data generation primers. Thus, it is possible to include these libraries as a small portion of an already scheduled run.

You design your PCR primers as usual. BUT you will use them as probes. You will need to:

• Convert the right PCR primer into its reverse complement. This becomes the right probe.
• Designed for amplicons under 250 bases.
• Create overlapped regions are needed if your region of interest is longer than 250 bases. Each probe pair hybridizes to one strand (not both sides like PCR). Overlapped pairs are on opposite strands.
• Position the overlaps to have redundancy in critical bases.
• Add the PCR1 and the PCR 2 regions to be compatible with the NGS for CE kit.
  NGS for CE sequencing. (A) A probe pair flanking the region of interest consisting of one left hand and one right hand (5’ phosphorylated) probe sequence (LHS and RHS) is added to denatured sample DNA. Post hybridization, the addition of polymerase and ligase extends successfully hybridized LHS and ligates the extended LHS to successfully hybridized RHS. (B) The ligated products are template for sample index PCR. The PCR primers (diagonal arrows) contain a universal priming site (blue diagonal, PCR1 and PCR2), a sample index sequence (red diagonal) unique to each sample, and the sequences required for NGS data generation on an Illumina platform (green diagonal).

You have the extracted DNA. This DNA is hybridized with your probes. The same probes are used with all the samples. After the hybridization, extension and ligation step, the samples are mixed with the Eureka Genomics samples indices. A PCR reaction adds the sequences that are needed for NGS data generation and the sample index. Post – PCR all the samples are pooled. The pooled library is cleaned up and the library quality is checked and passed onto NGS QC and data generation. Post-sequence data generation, the EG de-indexing software puts the sequences for each sample in a separate file.

The kits are available in six (6) sets of non-overlapping 384 sample indices and contain the polymerase.