Targeted sequencing, which allows you to focus on sequences and region of interest, encapsulates two different techniques: a broad, target capture via a hybridization method or a more focused, PCR-based amplicon panel design. Among its most notable benefits compared to other methods in NGS such as WGS or WES, is the fact that because less genomic sequence is being interrogated, only target regions of interest are sequenced. This means that by focusing the sequencing real estate of the flow cell to only regions of interest, genomic sequences can be enriched anywhere from 100 to a 1 million times compared to DNA-sequencing. This is critical for some applications where the typical 10x or 100x coverage obtained by DNA-sequencing or even large exome-sequencing may not be sufficient, such as for the detection of low level somatic variants that may require 3,000x coverage or more to be detected at a frequency of 5% or less. Similarly, great confidence can be got with deep sequencing via NGS than Sanger sequencing for these low-frequency variant calls.
PerkinElmer offers a complete line of NEXTFLEX® library preparation kits to meet your targeted sequencing needs.
Amplicon panels, which excel over target-capture hybridization methods that cast a broader net into the genome, are the technology of choice for small target panels ranging anywhere from 5 – 50 kb of sequence. They are also much cleaner, more rapid, and simpler to carry out on the bench when compared to any method requiring hybridization due to the fact that it is PCR-based. They also tend to be the most cost-efficient for sequencing as it requires the least amount of sequencing per sample for most applications, such as in the detection of germline mutations that only typically require coverage of approximately 50x – 500x. Similarly, as a PCR-based technology, different tactics can be employed that would not be viable using other sequencing methods to tackle the resolution of difficult genomic regions, such as those with high AT-rich or GC-rich sequences and paralogues/pseudogenes. Amplicon panels can sometimes be used in the context of targeted re-sequencing due to their ability to interrogate regions that may have been missed or insufficiently covered by WGS or WES. Their utility as an alternative option to reflex Sanger sequencing of problematic regions has also been adopted by some in the field as well.
Hybrid capture-based enrichment is one of two commonly used strategies for targeted sequencing methods. While amplicon panels use amplification of the region of interest via primer pools, hybrid capture-enrichment involves capturing regions of interest using hybridization probes. A much larger gene content and profiling is possible with this approach, with the given understanding that a longer turnaround time and more involved hands on handling is required. Both array-based hybrid capture and solution-based target enrichment employ probes that capture and select for target sequences while washing away non-specific hybrids via a wash or purification by magnetic bead. Subsequent amplification may take place prior to sequencing. Most common applications include SNP, indel, and CNV detection.
16S and 18S rRNA sequencing are useful and cost-effective NGS tools for microbial sequencing for metagenomic studies comparing different species of bacteria or for eukaryotic cells (18S ITS) present in a given sample without the need for cultures. 16s rRNA is a widely used gene marker for genus and species identification in bacteria and archaea for its minuscule rate of substitution in the highly conserved sequences outside of the hypervariable regions. 18S rRNA is routinely investigated in the phylogenetic study of fungi as it contains more hypervariable domains than 16S. The speed and economy associated with amplicon sequencing is maximized with this approach catered to population genetics and microbial profiling for interrogating the phylogeny and taxonomy of samples from complex microbiomes.
For research use only. Not for use in diagnostic procedures.