The right answers to frequently asked questions
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How do I provide target regions for enrichment?
The entire target sequence has to be provided
in FASTA or multiple FASTA format. For non-human samples the
minimum target size is 200 bp. All human target samples of <200
bp will be extended to 200 bp on the basis of the human genome
sequence (NCBI data at UCSC). Your target enrichment sequence may
consist of a continuous region or it may consist of independent
regions (e.g. exons). Total length of target sequence is given by
the array layout chosen.
What is the delivery time?
It depends on the size of your individual project and the sequencing technology applied.
Please refer to your individual quote.
What kind of quality controls do you perform before starting the sequencing of enriched samples?
The sequence capture techniques (on array or in-solution) contain built-in control probes.
We perform quality control on an amplified pool with qPCR to ensure system performance.
Can I perform sequence capture on organisms other than human?
In principle customised arrays can be designed for any other organism where reference sequences are available.
Is it possible to enrich repetitive regions?
Repetitive regions are not covered by the NimbleGen enrichment. They can not be sequenced.
However, repetitive regions that are flanked by unique regions are captured and be sequenced.
How much sample material do you need for sequence capture?
For sequence capture you have to provide us with at least 5 µg genomic DNA. NimbleGen Sequence Capture methods do not rely on amplification before hybridisation. Therefore potential sources of bias are excluded.
What regions can be captured by Roche NimbleGen Sequence Capture methods?
The customer can specify any region of interest where reference sequences are available, e.g. large contiguous genomic regions, for instance, whole chromosomes or non-continuous regions like specific exons or whole exomes of interest.
Who owns the design of the Roche NimbleGen Sequence Capture arrays?
Designs created for Roche NimbleGen arrays are property of Roche NimbleGen.
Are the array design data stored for further experiments?
Each specific customer capture design will be stored at Roche NimbleGen. You may order the same design in the future without additional design fees.
Is it possible to reuse sequence capture arrays?
Roche NimbleGen Sequence Capture arrays can not
How is sequence capture in solution performed with NimbleGen products?
- The SeqCap EZ oligonucleotide pool is made against target regions of the genome.
- A standard shotgun sequencing library is created from genomic DNA.
- The sequencing library is hybridised to the SeqCap EZ oligonucleotide pool.
- Streptavidin beads are used to pull down the complex of capture oligonucleotides and genomic DNA fragments.
- Unbound fragments are removed by washing.
- The enriched fragment pool is amplified by PCR.
- The success of enrichment is measured by qPCR at control loci.
- The end product is a sequencing library enriched for target regions and ready for high throughput sequencing.
What is the general workflow of a customer definded targeted resequencing project with Eurofins Genomics?
- The customer selects an established exome design or any desired genomic target regions for enrichment.
- We handle oligonucleotide design, production and data transfer.
- We enrich genomic samples and prepare the sequencing library.
Enriched samples can be sequenced with the Illumina HiSeq 2500.
- Bioinformatic analysis includes mapping of targeted re-sequencing data to a reference sequence, SNP analysis, quality statistic, coverage overview and other statistical analysis.
Specific requests can be addressed with a customised bioinformatics project.