NimbleGen Sequence Capture
Guidelines for Working with
Developer Products
Introduction
NimbleGen Sequence Capture products, including microarrays and solution-based SeqCap EZ probe
libraries, are tested and validated for use with human genome reference sequences (e.g. hg18, hg19) and
human DNA samples. However, many researchers are interested in developing their own custom
applications and protocols for use with the core Sequence Capture technology and materials. Some
examples include the following:
 Sequence Capture of genomic DNA targets from non-human, traditional genetic research
organisms (e.g. mouse, zebrafish, Drosophila, C. elegans, maize)
 Sequence Capture of genomic DNA targets from non-human organisms with less wellcharacterized genomes (e.g. cetaceans, poplar trees, mosquitoes)
 Sequence Capture of non-nuclear nucleic acid targets from humans or other organisms (e.g.
cDNA, mitochondrial DNA, chloroplast DNA, viruses)
 Sequence Capture of human genomic DNA targets using probes designed from non-reference
sequences (e.g. individual genome sequences)
 Sequence Capture of nucleic acid targets of any type using non-standard capture probe design
criteria requested by the researcher
To accommodate the needs for these custom applications from researchers, Roche NimbleGen offers the
following Developer products:
 Sequence Capture Developer 385K/2.1M Arrays
 SeqCap EZ Developer Libraries
Because protocols for use in non-standard applications like those listed above have not been tested by
NimbleGen researchers, we cannot provide users of Developer products with the same performance
assurances or level of technical support that we can for customers who use our standard Sequence
Capture products in fully validated applications.
To help address the needs of the broader scientific community, this document provides some general
guidelines intended to help Developer users achieve better results in their novel research applications.
Recommendations are provided for the following phases of a Sequence Capture experiment:
 Selecting targets and designing probes
 Performing Hybridization
 Performing Enrichment QC by qPCR
For life science research only. Not for use in diagnostic procedures.
Selecting Capture Targets and Designing Probes
The most important factor in designing a successful Sequence Capture Developer experiment is the
quality of the input sequence used to select the target and capture probes. Even when working with a
well-characterized dataset like the human genome, there are still gaps in the reference assembly and
regions that are subject to hypervariability and copy number variation that can introduce problems at
the design stage. These problems increase significantly when creating designs for species where the
amount and quality of the beginning sequence data are much lower.
NimbleGen can design Sequence Capture Developer probes from a complete genome sequence or from
incomplete sequences such as genome contigs, genome scaffolds, and even expressed sequence tag (EST)
and mRNA datasets. However, the ability to determine whether any given probe is contiguous along its
length with the actual genome target, or whether a probe is targeting a single copy sequence rather than
a repetitive element, can be compromised when using such data. Although we do not allow probes from
multiple species to be included on one design, when the sequence of the targeted species is minimal it is
sometimes possible to use the more complete sequence of an evolutionarily related species, if available,
to determine whether probes selected for the targeted species are likely to be unique or repetitive.
One approach that has been used to assist in the Sequence Capture Developer design process is to
perform a preliminary Comparative Genomic Hybridization (CGH) experiment targeting the region
intended for capture. The results of the CGH experiment can provide useful data on the hybridization
properties of individual probes and permit design modifications before attempting the capture
experiment. Similarly, one may consider an iterative design process in which data from a pilot Sequence
Capture experiment can be used to create an empirically optimized second-generation design. If you are
considering either option, it will be important to discuss your plans in advance with NimbleGen
scientists so that they can provide additional recommendations to facilitate the process. When the
planned Sequence Capture Developer experiment entails using a previously untested species or capture
design, or the input sequence used to select the target and probes is suspect or minimal, it is always
recommended that an initial, small scale, pilot experiment be performed before proceeding with larger
scale studies.
Performing Hybridization
The inclusion of human Cot-1 DNA as a blocking reagent in a human sequence capture (microarray or
SeqCap EZ probe library-based) experiment reduces the capture of non-targeted sequences. Part of this
reduction is likely due to the blocking of non-specific DNA binding by the glass (microarray) or the
biotin/streptavidin (SeqCap EZ probe library) complexes, but the remainder is due to competitive
inhibition of secondary capture. Secondary capture occurs when a capture probe hybridizes to its
intended target on a sample library DNA fragment, but a different part of that captured fragment
contains repetitive sequences. Those repetitive sequences, since they are single-stranded, can act as
secondary capture ‘‛probes‛ and subsequently hybridize to any DNA fragment in the sample library
containing a homologous repetitive sequence.
For Sequence Capture Developer experiments targeting species for which the appropriate Cot-1 DNA is
commercially available (e.g. mouse, rat, cow), we recommend substituting that species’ Cot-1 DNA in
place of human Cot-1 in the hybridization reaction. For experiments targeting species for which the
appropriate Cot-1 DNA is not commercially available, it is possible to prepare your own custom Cot-1
DNA. Examples of published methods include the following:
 Britten, et al. (1974) Analysis of repeating DNA sequences by reassociation. Methods Enzymol.,
29, 363–418.
 Zwick et al. (1997). A rapid procedure for the isolation of Cot-1 DNA from plants. Genome, 40(2):
138–142.
In some cases, it may be possible to obtain adequate results by using Cot-1 DNA derived from a closely
related species that shares similar repetitive sequences. The necessity of including a species-specific Cot1 blocker in the hybridization reaction will vary depending on the frequency and type of genomic
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NimbleGen Sequence Capture: Guidelines for Working with Developer Products v1.0
repetitive elements in the genome of the species targeted for capture. The absolute amount of Cot-1
DNA that is required to reduce non-specific or secondary capture in a Developer experiment may differ
from the amount of Cot-1 recommended in human sequence capture protocols due to the frequency of
different repeat types, their hybridization kinetics and overall genome complexity. In some cases, it may
be sufficient to use a general nucleic acid blocker (e.g. sonicated yeast genomic DNA, herring sperm
DNA) to reduce non-specific capture, or no blocker at all.
Performing Enrichment QC by qPCR
This section provides general guidelines for developing novel quantitative PCR (qPCR) assays to
estimate Sequence Capture enrichment from non-human genomes using custom Sequence Capture
Developer Arrays or SeqCap EZ Developer probe libraries. Use these guidelines in combination with the
NimbleGen Sequence Capture User’s Guide that is most similar to your intended application.
qPCR provides a fast, reliable and low-cost means of estimating the success of a Sequence Capture
experiment prior to sequencing captured DNA fragments. All NimbleGen Sequence Capture arrays and
SeqCap EZ probe libraries include capture probes targeting a set of human control loci. Following a
capture experiment, standardized qPCR assays are used to compare the pre-captured amplified sample
library with the captured amplified sample library to estimate the relative enrichment at these control
loci and to detect potentially failed capture experiments. Because the utility of this particular set of
control loci and qPCR assays is limited to experiments with human samples, we strongly recommend the
design and implementation of similar, species specific, qPCR assays and controls for use in custom
Developer experiments by following the general guidelines provided below:
Step 1. Identifying Candidate Control Loci for Your Species
1. We recommend developing at least 4 control loci/qPCR assays for use with NimbleGen Sequence
Capture Developer experiments. Use of greater than 4 control loci may produce more accurate and
consistent results, but may be impractical for low-cost and/or high-throughput applications.
2. From the entire target for which capture probes were successfully designed, choose a subset that is
representative of the larger target in both size and G+C content. Choose targets that are less likely
to be affected by copy number variation or population genetic heterogeneity.
3. Keep in mind that of the loci initially chosen, not all may have the optimal probe coverage required
for use as a control locus, or the sequence that permits the design of effective qPCR assays. You may
have to begin with an initial set of >20 targets to ultimately obtain 4 useful control loci.
Step 2. Developing qPCR Assays Specific to the Candidate Control Loci
1. Design qPCR assays for each of the selected loci. Either intercalating (e.g. SYBR Green) or probebased (e.g. TaqMan® probes) assays may be developed, but the inherent advantages and
disadvantages of both types should be considered. Use similar assay design parameters (e.g. primer
melting temperatures, product lengths) to ensure that the resulting assays can be performed under
the same reaction conditions.
2. Test the qPCR assays. The assays should be:
 Specific (i.e. produce a single peak at the predicted melting temperature)
 Consistent (i.e. produce similar results in replicated reactions and experiments)
 Sensitive (i.e. able to detect the target at a wide range of concentrations)
We recommend testing the qPCR assays with genomic DNA of all the species and substrains that
will be used in planned Sequence Capture experiments. The amount of DNA to use as template will
vary depending on genome size and complexity, and should be determined empirically. For
NimbleGen Sequence Capture: Guidelines for Working with Developer Products v1.0
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developing mammalian qPCR assays, we recommend testing each assay using 0.04 ng, 0.2 ng, 1 ng,
5 ng, and 25 ng of mammalian genomic DNA as template, in triplicate reactions.
3. Use the resulting data to rank the assays by specificity, sensitivity, and efficiency of each qPCR assay.
The PCR efficiency number is used later in calculations to determine target enrichment (for more
details, see the chapter on ‚Measurement of Enrichment Using qPCR‛ in one of the NimbleGen
Sequence Capture User’s Guides). Discard any assays that generate multiple products or show a low
efficiency (<1.5).
Step 3. Evaluating Candidate Control Loci Assays
1. Candidate control loci assays should be functionally tested by performing a Sequence Capture
experiment and comparing the qPCR enrichment estimates directly with sequence from the
enriched library. Refer to the chapter on ‚Measurement of Enrichment Using qPCR‛ in one of the
NimbleGen Sequence Capture User’s Guides for general instructions on performing control loci
assays, substituting your candidate control loci for the standard set of human loci. Assays that
predict high or low enrichment are both useful as long as the prediction is confirmed by the
sequence analysis.
Note: A Sequence Capture enrichment experiment may be simulated for the purpose of developing
QC-assays, by adding known concentrations of PCR products from the candidate control loci into
genomic DNA at different ratios.
2. Select a set of control loci that are likely to well represent the larger set of target loci and have a
distribution of capture efficacy (i.e. some loci enrich poorly, some loci enrich very well, and most
loci enrich somewhere in between). This QC assay set should be tested in multiple Sequence
Capture experiments to generate dependable performance data.
Alternative Method
06465528001  11/11
One possible alternative to developing qPCR assays using the approach described above is to use the
Roche Universal ProbeLibrary. The probes in this library were developed from transcribed sequences
and intended primarily for use in gene expression analysis, but they can be adapted for use in qPCR
assays to estimate Sequence Capture enrichment. For more information about the Roche Universal
ProbeLibrary, go to https://www.roche-applied-science.com/sis/rtpcr/upl/ezhome.html.
For life science research only. Not for use in diagnostic
procedures.
NIMBLEGEN, SEQCAP and TAQMAN are trademarks of Roche.
Exiqon and ProbeLibrary are registered trademarks of Exiqon A/S, Vedbaek,
Denmark.
SYBR is a registered trademark of Molecular Probes, Inc.
All other product names and trademarks are the property of their respective owners.
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Published by
Roche NimbleGen, Inc.
504 S. Rosa Rd
Madison, WI 53719 USA
© 2011 Roche NimbleGen, Inc. All rights reserved.
NimbleGen Sequence Capture: Guidelines for Working with Developer Products v1.0