FAQ – Sequence Enrichment Assay
- What are the DNA sample requirements for the Sequence Enrichment Assay?
- Is the Sequence Enrichment Assay compatible with whole-genome amplified (WGA) DNA?
- How much target sequence can be enriched with a single primer library on the RDT 1000?
- How many droplets are created for each sample with the Sequence Enrichment Assay?
- How much genomic DNA is contained in each droplet?
- How many individual PCR reactions are generated for each primer pair?
- How many copies of each PCR amplicon are generated in the droplets?
- What is the size range of amplicons generated with the Sequence Enrichment Assay?
- What is the range of %GC for PCR amplicons with the Sequence Enrichment assay?
Q – What are the DNA sample requirements for the Sequence Enrichment Assay?
A – RainDance recommends using only high-quality Genomic DNA for the Sequence Enrichment Assay. The purity of each sample should be determined by evaluating the ratio of the O.D. measurement at 260 nm and 280 nm (A260/A280). Samples should have an A260/A280 >1.8. The DNA should also be of high molecular weight and should generate a single band on a 1% agarose gel that is approximately 10kb or higher.
Q – Is the Sequence Enrichment Assay compatible with whole-genome amplified (WGA) DNA?
A – RainDance does not currently support the use of WGA DNA in the Sequence Enrichment Assay. However, we are developing protocols for handling WGA DNA with the expectation of supporting WGA by the middle of 2009.
Q – How much target sequence can be enriched with a single primer library on the RDT 1000?
A – At present, the RDT 1000 supports custom primer libraries containing up to 1,536 unique prime pairs. The total amount of target sequence represented will vary depending on the nature of the specific target sequences. The typical target sequence for exons is 384 Kb. Typical target sequence for contiguous regions is 192 Kb. Custom order primer libraries containing up to 4,000 primer pairs will be available in July 2009.
Q – How many droplets are created for each sample with the Sequence Enrichment Assay?
A – Each sample processed on the RDT 1000 using the Sequence Enrichment Assay results in approximately 1 million droplets.
Q – How much genomic DNA is contained in each droplet?
A – Each template mix droplet contains on average 1.5 pg of genomic DNA, which is equivalent to approximately 43% of a haploid human genome.
Q – How many individual PCR reactions are generated for each primer pair?
A – The number of PCR reactions generated for each primer pair varies depending on the number of primer pairs in the primer library. The number of "PCR positive" droplets is estimated based on statistical sampling of the randomly mixed primer library assuming a 43% probability that the PCR droplet will contain the target sequence corresponding to the primer pair merged with the template mix droplet. Primer library of 384 primer pairs will result in a minimum of 447 PCR reactions per primer pair. A primer library of 1,536 primer pairs will result in a minimum of 112 PCR reactions per primer pair. A primer library of 4,000 primer pairs will result in 43 PCR reactions per primer pair.
Q – How many copies of each PCR amplicon are generated in the droplets?
A – Typical enrichment experiments result in a minimum of 250 ng of PCR product. This equates to an average of 2,500,000-fold amplification per droplet assuming an average amplicon length of 500 bp.
Q – What is the size range of amplicons generated with the Sequence Enrichment Assay?
A – The current range of amplicon size for the Sequence Enrichment assay is 150-600 bp. The actual size range will vary depending on the sequencing platform of choice (long read vs short read).
Q – What is the range of %GC for PCR amplicons with the Sequence Enrichment assay?
A – The RainDance primer design process limits the GC content of amplicons to 30-65%.
