The RareSeq™ workflow begins with basic extraction of genomic DNA (gDNA). The gDNA is quantified by a fluorescence-based method, such as Qubit, and quality is assessed by running an aliquot of the sample on a gel to ensure that the DNA is intact and not sheared. If a compact single band is observed with little or no smearing, the gDNA is of good quality.

Based on the quantification results, 250ng of each sample is used as the starting material for the library prep. Next, we hybridize the oligo pool to the sample. This oligo pool contains upstream and downstream oligos that are specific to the regions of interest. After removing unbound oligos, extension and ligation occurs. Extension and ligation connect the hybridized upstream and downstream oligos. A DNA polymerase extends from the upstream oligo through the targeted region, followed by ligation to the 5′ end of the downstream oligo using a DNA ligase. The result is the formation of products containing the targeted regions of interest flanked by sequences required for amplification.

The gDNA now has specific oligos that have been hybridized, extended and ligated to target regions. For example, in the case of the TruSight® Myeloid Sequencing Panel, there are 568 amplicons representing 54 genes. The next step includes the addition of both the i7 index and our 16bp RareSeq™ UMI (unique molecular index) through PCR. The i7 index allows for sample pooling prior to sequencing. At the end of this step, PCR products are bead-purified.

A key aspect of the RareSeq™ workflow is quantification using droplet digital PCR (ddPCR). Using the product from the previous step, serial dilutions are made with the least concentrated is quantified by ddPCR. In ddPCR, the sample is partitioned into 20,000 nanoliter-sized droplets. This partitioning, droplet generation, enables the measurement of thousands of independent amplification events within a single sample. The amplification reaction is then read based on fluorescence and statistically analyzed to determine the concentration of the original sample.

Based on the ddPCR, samples are normalized such that each sample has the same number of starting molecules. Normalized samples are then PCR amplified using P5 and P7 primers, and the PCR products are bead-purified.

The final libraries are run on the Agilent TapeStation to confirm the quality of the DNA. Libraries are then pooled for sequencing. The desired level of sensitivity determines the number of samples that can be pooled in a single lane. For example, a sensitivity of 1:5000 requires fewer reads per sample than a limit of detection of 1:10,000. Therefore, more samples can be pooled if 1:5000 is desired compared to 1:10,000. After pooling, a final concentration is determined using a fluorescence-based method, and the libraries are sequenced on an Illumina sequencing platform.

Once sequencing data is received, the samples are demultiplexed and consensus reads are assembled.  Samples are clustered into read families based in the UMIs.  This is where RareSeq™ sets itself apart from other error correction techniques as errors introduced during PCR and NGS are then corrected by comparing read families so that variant allele frequencies (VAF) as low as 0.0002-0.0001 can be identified.

TruSight® is a registered trademark of Illumina, Inc. and is used without permission. No challenge to the rights of Illumina is intended or implied.


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