Custom Capture Sequencing

Custom Capture Sequencing

With a complete sequencing platform and a strong technical team, CD Genomics can provide professional custom capture sequencing services to research teams and scholars working in genomics and other related fields. For any sample type, parameter requirement, or index sequence, we can provide a quality service.

What Is Custom Capture Sequencing?

Custom capture sequencing (CCS) is the selective sequencing of specific (targeted) regions of interest in the genome to discover rare or common variants associated with a disease or phenotype. The strategy of concentrating the sequencing needed within a targeted region of the genome can be extremely cost and time-effective, and CCS can be performed either by targeting exons of selected genes or by sequencing entire genomic regions. The cumulative size of the target region of interest typically ranges from approximately 500 kb to 50 Mb.

How Is CCS Performed at CD Genomics?

The premise of choosing a custom capture service is that you have identified one or more areas of interest. CD Genomics staff will use the information you provide to develop a custom sequencing protocol, designing oligonucleotide probes for hybridization capture or multiplex PCR primers that can be used to enrich target regions of the genome. CD Genomics will discuss this with you in detail before finalizing the plan. Note that custom capture projects typically include 24-576 samples.

What Material Should You Send for CCS?

We need a minimum of 150 ng of highly-purified genomic DNA (0.5 μg preferred) in a volume of 50 μL or less for CCS. Samples should be submitted in 1.5-1.7 mL microfuge tubes or 2 mL screw cap tubes. Please DO NOT send samples in 0.5 or 0.2 mL tubes. To ensure that each sample is uniformly pure and free of infectious agents, we strongly recommend that all DNAs be phenol:chloroform extracted before submission. A simple protocol is available from CD Genomics.

How Should the DNA Be Qualified?

You must submit an image of an analytical agarose gel or a trace as evidence the DNA is of good integrity and the appropriate molecular weight for the sequencing approach. We highly recommend Qubit for quantitation of the DNA sample, since it uses a double-strand DNA-specific method. The UV absorption method is not an ideal choice for quantification and it can greatly overestimate the concentration of DNA.

How Long Are the Reads for CCS Analyses?

Typically, NISC generates read lengths of 150 bases on a NovaSeq 6000. Paired-end reads generate a total of 300 bases of sequence (150 b from each end) from each fragment in the library.

How Many Reads Are Required for CCS Analyses?

We target the number of reads that are calculated to produce an average read-depth of greater than 100× coverage.

How Are Variants Called?

After sequencing is completed, we process this information using a probabilistic Bayesian algorithm. The probability of each possible genotype is calculated from the obtained sequence data and an MPG (most likely genotype) score is given, which is considered accurate when the MPG is 10. We have done validation that genotypes with MPG scores of 10 or higher show >99.89% agreement with the SNP microarray data.

What Data Are Returned by CD Genomics?

We return data containing all identified variants and genotypes of all sequenced samples, as well as gene location (5'UTR, 3'UTR, coding synonymous, nonsynonymous, or stop, splice site, or intron).

Custom Target Panel

• Standardized sequencing protocols to avoid common problems during sequencing
• Customizable sequencing protocols
• Efficient, reproducible, and scalable high-throughput sequencing services

Our Workflow

CD Genomicsis a leader in sequencing services, and as a global company with a specialized sequencing technology platform and technical team, we value collaborative interactions and provide high-quality custom capture sequencing services. If you are interested in custom capture sequencing services, please contact us today for expert support!

References

• Sims, D.; et al. Sequencing depth and coverage: key considerations in genomic analyses. Nat Rev Genet. 2014, 15(2): 121-132.
• Teer, J.K.; et al. Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res. 2010, 20(10): 1420-1431.
• Teer, J.K.; et al. VarSifter: visualizing and analyzing exome-scale sequence variation data on a desktop computer. Bioinformatics. 2012, 28(4): 599-600.

For research use only, not for any clinical use.