Targeted Sequencing - FAQs

Targeted Sequencing

Additional Information

Product Literature

FAQs

Answers - General

Why sequence DNA?

Sequencing DNA gives us the ability to massively digitize the genetic codes and discover SNPs and other forms of mutations in the genome. This can increase our understanding, diagnosis, treatment and prevention of disease.
Comparative resequencing – (analyzing variations across and within species or samples subjected to different treatments) enables genome-wide association studies and provides a more comprehensive understanding that can enable development of personalized medicines and clinical diagnostic.
De novo sequencing has applications for medical, pharmaceutical, forensics, identification, defense, and basic research.
Transcriptome sequencing – gene expression profiling, exon sequencing, trans-splicing RNA sequencing, identification of novel transcripts, splice variant detection, transcriptional mutation, miRNA, small RNA, non-coding RNA, etc.

What is target-specific sequencing?

Sequencing only a part of the original sample by enriching - or depleting certain types of sequences. Target specific sequencing allows mainly the sequence(s) of interest to be determined.

What are the advantages of "targeted sequencing"?

Target-specific selection from a sample for a defined region by length or sequence, such as cancer suspect regions or known cancer genes, SNP regions, specific regions of interest (of unknown sequence).
Target-specific selection of RNA sequences, such as sets of coding genes known from databases, such as RefSeq mRNA or Ensembl gene, previous experiments, specific disease or pathway related RNAs, novel sets of RNAs, miRNAs, and biomarker RNAs.
General use of the capture probes on microarray to enrich target sequences.
Depletion of non-targeted sequences, such as depletion of those interfere with sequencing reactions, high abundance repeating sequences.
Reduction of the complexity in samples to enable multiplexing, high coverage, and high dynamic range or in-depth sequencing.
Obtain cleaner sequencing results to simplify data analysis and reduce time to final results.
Overcome the limitations of multiplex and/or long-range PCR. Take advantage of huge time and cost savings.

How is target-specific selection achieved?

Sequence selection can be achieved by hybridization using complementary capture probes or by sequence-specific ligand molecules such as DNA/RNA binding molecules including small ligands, drug molecules, proteins/antibodies/peptides, aptamers, etc which bind to the sequence of interest.

On-Chip or Microarray-Based Target Selection

Capture probes are immobilized on surface for hybridization. Samples are hybridized to a microarray containing capture probes designed to target specific regions of interest in any genome (or transcriptome). Undesired sequences are washed off and captured target sequences are recovered by eluting from the chip. These selected target sequences are ready for high-throughput sequencing or they can be further processed.

In-Solution Target Selection

Capture probes can be used for solution hybridization. One method involves using oligonucleotides (OligoMix^®) in solution as capture probes which are designed to target specific genomic/sequence regions of interest. After hybridization with a sample, magnetic beads are added and the capture probes are affinity linked to the beads. The captured target sequences are separated from other sequences by washing the beads after which the target sequences can be recovered. Another method involves using oligonucleotides (OligoMix^®) immobilized on beads.

Is this service compatible with all next-gen (high-throughput) sequencing platforms?

Yes, samples enriched (or treated for depletion) by our target selection processes are compatible with Roche/454 FLX, ABI SOLiD, Illumina(Solexa) GA sequencers.

Which regions can be targeted with this technology?

Almost any genomic regions of any species, and all known RNA transcripts can be targeted by this technology as long as capture probes can be designed.

What is the role of specificity in target-specific selection?

In target-specific selection for sequencing, often the goal is target sequence enrichment. Frequently it is more important to capture the highest possible percentage of the sequences of interest while sacrificing non-specific capture. In comparison, microarray hybridization requires high specificity for hybridization probes in order to obtain accurate signal reading for the genes analyzed. Through capture probe design, the degree of specificity can be varied according to experimental requirements. Capture probes can be designed to be as specific as microarray probes when this is required.

What sequence information do you need? How specific does the region interest need to be described?

Of course the more information provided, the better. However, at least accession number(s) for the original sequence(s) should be provided so that the selection probes can be designed from that information. There is a one-time charge for design of capture probes. If you prefer, you can design your own capture probes and at your request, we can work with you to optimize the probe design.

Sample Preparation

What types of samples are amenable to target-specific selection?

Genomic DNA, PCR libraries (amplified DNA), or RNA (polyA, miRNA, small ncRNA, others)

What are the sample requirements?

Genomic DNA –Use of the Qiagen Kit for DNA extraction is recommended. Samples should contain at least 20 µg genomic DNA with an OD260/280 ratio of 1.8 to 2.0.

Amplified DNA - Samples should contain at least 8 µg PCR product. The quality of the sample should be determined with a Bioanalyzer or a gel to confirm length <1,000bp.

mRNA – Use of the Qiagen RNeasy kit for total RNA extraction is recommended. Samples should contain at least 10 µg with an OD260/280 ratio of 1.8 to 2.0. Check the RNA quality with a Bioanalyzer, or a 1-1.5% agarose gel. High quality RNA will show a 28S rRNA band at 4.5kb that should be twice the intensity of the 18S rRNA band at 1.9kb. Excessive smearing indicates degraded RNA.

Small RNA –Use an RNA extraction kit that will retain small RNA in the total RNA sample such as the Qiagen miRNeasy kit is recommended. Samples should contain at least 10 µg with an OD260/280 ratio of 1.8 to 2.0. Check the RNA quality with a Bioanalyzer, or a 1-1.5% agarose gel. High quality RNA will show a 28S rRNA band at 4.5kb that should be twice the intensity of the 18S rRNA band at 1.9kb. Excessive smearing indicates degraded RNA.

Sample Submission

What forms are required for sample submission?

Please complete a sample submission form prior to sending your sample to us. We request that you email the form to us prior to sending the sample AND place a copy of the form in the package with your sample(s).

We cannot begin work on your project without a PO Number or Credit Card Number.

Please see our sample submission page for further information.

How do I pack and ship my sample?

Please transfer your sample to a 1.5ml microcentrifuge tube for shipment (0.5 ml or smaller PCR tubes can crack when frozen).

Use only parafilm if you want to seal the tubes or hold them in a rack. Please don't use tape (it will crack when frozen).

Pack the sample with dry ice in a thermal insulated shipping box. Ship by overnight carrier for delivery the next day.

Note: Do not ship samples on Friday as they will sit over the week-end and deteriorate. Wait until the following Monday to ship the package for Tuesday delivery.

Where do I send my sample?

Mail your package to:                       Attn: Targeted Sequencing Sample
                                                           LC Sciences
                                                           2575 West Bellfort Street Ste 270
                                                           Houston, TX 77054

Can I send LC Sciences a sample from overseas?

Yes, please download a copy of our International Shipping Instructions.

Can samples be returned to me?

Yes, we can return unused sample to you upon request at the time of order. Please note that samples will not be stored after your experiment has been completed unless you specifically instruct us to do so. We will store the sample for a maximum of one year after your experiment has been completed. We can not be responsible for sample degradation or loss during mailing or storage.

Deliverables

How long will it take to get results?

We can generally have product back to you about 4-6 weeks from the date we receive your sample. We also offer a 3 week rush service.

How much final product will I receive?

You will receive 1 µg of amplified DNA which can be used directly for high-throughput sequencing.

What information will I receive with the final product?

You will receive a report containing capture probe sequences, hybridization images, and data analysis.

If optional sequencing service is selected - You will receive a final report for the sequencing service, in which a special section is included for analysis of the capture coverage and efficiency.

Should microarray experiments be considered as a next step?

Sequencing provides massive data from which sub-sets of sequences can be identified which are associated with sample conditions. Microarray is an effective, fast and cost-effective tool for the next step in your research. For our customers, we provide a free service for next step array probe design based on the sequencing results.

For other questions please send us an email at service@lcsciences.com or call us at 1-888-528-8818

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