Flex-Seq FAQs

Once you have ascertained which SNPs are to be further interrogated via your linkage and association studies, Flex-Seq can be used as a targeted approach for downstream screening. It should be noted that a pilot GBS may not provide sufficient information for a Flex-Seq project. Please contact technical and applications support regarding the options for your specific project.

While we no longer offer array genotyping services, we have improved our support to include sequencing-based genotyping as a replacement strategy. By working with our expert team to design a custom SNP panel specific to your breeding or genotyping program, you avoid the wasteful coverage of irrelevant targets in off-the-shelf arrays while gaining insights and new information specific to your strains. Sequencing with Flex-Seq can not only reveal whether a SNP of interest is present but also provides additional information on the surrounding sequence potentially revealing valuable insight and identifying new markers.

• Flex-Seq targeted genotyping by sequencing service to screen tens of thousands of markers on an industrial scale.
• GBS genotyping by sequencing is the method of choice for genome wide SNP discovery without prior knowledge of the genome sequence and is the ideal first step when moving from array genotyping without a well-defined reference genome.
• High quality DNA extraction service can start with a wide variety of starting materials and includes a convenient collection kit. Extracted DNA can seamlessly flow into sequencing projects or be shipped on to array service providers as preferred.

Yes, we are able to accommodate very large project requirements, but we do not offer pre-validated SNP panels. SNPs to be included are decided on by the customer. We do have a number of Flex-Seq panels designed for a variety of species, which you may use as a starting point to design your ideal SNP solution.

An all-inclusive Flex-Seq service includes

• BioArk Leaf Collection Kit / BioArk Seed Collection Kit
• DNA extraction service
• Preparing of a sequencing library and sequencing
• Labour costs
• Bioinformatics (SNP genotyping)

Additional bioinformatics analysis may incur extra charges. We can also offer custom service packages, so please contact technical and applications support for further information.

The Flex-Seq algorithm software will design two probes which are used to target individual regions, often focusing on a single SNP. Probes are designed to flexibly bind on either side of the SNP, leading to greater ability to recover desired targets.

The Flex-Seq algorithm software will design two probes which are used to target individual regions, often focusing on a single SNP. Probes are designed to flexibly bind on either side of the SNP, leading to greater ability to recover desired targets.

The standard Flex-Seq service for known SNPs is 2x150bp read. Using this, we can screen for customer-identified SNPs/InDels within a 100-200 bp region. We can also provide custom services with different read lengths, so please contact technical and applications support for further options.

All SNPs identified for interrogation by Flex-Seq must achieve a minimum of 10x coverage (a minimum of 3 reads per allele, for a bi-allelic SNP), otherwise the target SNP is not assigned a genotype. If greater confidence is desired, an increased minimum coverage can be applied and this must be discussed prior to the commencement of the project. We will recommend that an appropriate level of multiplexing is performed in relation to the capacity of the sequencing run to ensure the minimum coverage per sample is met.

Yes, Flex-Seq can be used with CRISPR. Please be aware that only the SNPs identified in the design BED file which correlate with the reference genome will have probes designed for. Flex-Seq will generate sequence data for the full interval between the probes (often 150-250bp) and thus can be used to characterize CRISPER based mutation. Probe design will only be performed on predetermined regions; this means any true off-target SNPs will not be accounted for.

Yes, Flex-Seq can be used for both SNPs and small InDels and short repeats. For design the target interval (including any SNP, or small indel) should not exceed 30 base pairs. For insertions the length of the insertion should not exceed 30bp.

The issues that can cause Flex-Seq probe design failure are similar to those for conventional PCR (i.e. high/low GC content, low complexity regions, repetitive motifs flanking the markers). Flex-Seq is significantly more tolerant to polymorphism in the probe binding region, making this factor less of a concern. Therefore a “pre-filter” is included in all Flex-Seq assay design to deselect these regions in order to increase the success rate for downstream probe binding.

Yes, one major advantage of Flex-Seq over array-based technologies is the flexibility in the selection of SNPs. Should any SNPs fail at the design stage these can be exchanged for alternative SNPs prior to library generation.

We ask the customer to submit a greater number of SNPs than is desired (+10-20% recommended, +50-100% preferred) to aid oligonucleotide probe design. All submitted SNP sequences (which both pass and fail design) will be reported back to the customer for final selection before synthesis of the kits.

The approximate read length is 75 bp. We can also provide custom services with different read lengths, so please contact technical and applications support for further information.

Yes, all that is needed for Flex-Seq design is the location of the SNP/target relative to the reference genome. The allelic makeup of the SNP is not needed neither is information on flanking polymorphisms. Please note that you will also need to provide the corresponding reference genome.

Yes, reference scaffolds can be used. Another option would be to use a very closely related species. If either of these options is chosen, it is the responsibility of the customer to ensure that the SNP locations in the BED file match the SNPs in the species of interest.

All Flex-Seq projects require:

• a reference sequence (genomic or transcriptomic, or a reference scaffold). These can a provided as a link to a publicly available genome or submitted as a FASTA file.
• a list of SNP locations to be interrogated, submitted as a VCF, Excel or BED file.

Yes, however, the organism/species should be very closely related. It is the responsibility of the customer to ensure that the SNP locations in the polymorphism definition file match the SNPs in the species of interest. Note that differences between the reference genome provided and the genome sequenced may lead to lower-than-expected quality scores.

All Flex-Seq projects require:

• a reference sequence (genomic or transcriptomic, or a reference scaffold). These can a provided as a link to a publicly available genome or submitted as a FASTA file.
• a list of SNP locations to be interrogated, submitted as a VCF, Excel or BED file.

Generally, compared to other targeted genotyping methods, Flex-Seq is more tolerant of known or unknown SNPs in the probe binding region. However, the quantity and location of the variants can have a negative impact on probe hybridization and can lead to increased missing data.

When the flanking SNPs are unknown, and suspected to be high, we would recommend a trial study using the fragmented draft reference genome to assess the project feasibility. In terms of probe design, a mismatch that is located in the middle of the probe is typically acceptable.

Currently, we do not assign a score, but information is available on the estimated off-target hit count for each probe, and the number of successful probes for each target SNP. Further information may be available, so please contact technical and applications support for further details.

It is advisable to send at least 2x – 3x the number of candidates for the number of markers you wish you interrogate (e.g. for 1000 markers, please submit 2000 – 3000 candidates). This will increase the chances that a greater number of probes (i.e. >1000) will be generated in the first design iteration. Please note, design success is variable based on the candidates chosen and the genome of the organism.

No, these databases are regularly updated, so there may be minor variations between the customer sequence and the published sequence. Please provide a link to the exact version of the genome to be used as a reference sequence, to which the positions in the polymorphism definition file refer (e.g. GRCh38).

We gather summary statistics for QC purposes, but we currently do not offer any downstream statistical analysis for genotype comparisons of samples or groups of samples.

In brief, we perform the following:

• a pre-processing step to remove synthetic and low-quality sequence
• an alignment against the reference sequence
• a pileup at the targeted position
• a fixed-threshold calling to genotype the sample

We do not use a predefined Bio-IT platform. We have our own pipeline for mapping reads to the region of the SNP of interest, and the SNPs identified are available in a call-variant table. In addition, a report with the number of parameters of the project is also provided, and the raw data from the Illumina instrument is available.

Depending on the size of your project, your data will be sent to you, either on a USB memory stick, or hard drive. All FASTQ data will be returned along with a variant file with variant calls by position. If additional bioinformatics is desired, please discuss this with your dedicated Flex-Seq project manager, who will be assigned to you on the commencement of your project.

The data returned will be a .csv file with a quality score assigned to each SNP, which can also be in the format of the number of reads per allele. The customer will also get the raw data file from the Illumina instrument.

The "polymorphism definition file" is used to identify the position of the SNPs and insertions and deletions (indels) of interest, when compared against the submitted "reference genome". The polymorphism definition file can be submitted in BED, VCF or Excel file format.

Flex-Seq industry standard panels exist for maize, corn, sugarcane, coffee, cotton, cranberry, strawberry, blueberry, cranberry, sugarcane, Douglas Fir, Radiata Pine, cattle, and porcine. Successful projects have been run on many other species including poultry, watermelon, radish, melon, tomato, brassica and celery.

Yes, with the caveat that the SNPs identified allow for the distinction between the different genomes and as long as reference genome and SNP information/locations have been identified.

All Flex-Seq projects require:

• a reference sequence (genomic or transcriptomic, or a reference scaffold). These can a provided as a link to a publicly available genome or submitted as a FASTA file.
• a list of SNP locations to be interrogated, submitted as a VCF, Excel or BED file.

Please contact technical and applications support for further information if you have any queries about your organism. Please, also refer to the Flex-Seq guidance notes for details on sequence submission.

Unfortunately, as this is a diagnostic request, this is a project we would not be able to support. Also, the limiting factor would be the concentration of the target organisms. A process of filtration/concentration would need to be performed, and/or an amplification/culture step. By introducing amplification steps (e.g. pre-amplification PCR or a nested-PCR), bias may be introduced in terms of proportion of each target. In addition, culturing would only work on bacterial species and would not be applicable for non-cultivable organisms (e.g. certain species of parasites and viruses).

The QC of the submitted DNA prior to library preparation is part of the Flex-Seq service. However, we recommend DNA be extracted using methods that yield higher quality DNA, such as column, bead based, or C-TAB extraction to minimise factors such as secondary compounds/metabolites, chemical carryover from extraction, or excessive degradation. We discourage use of "crude" DNA extractions.

We recommend running 5 – 10 ng per sample of DNA using a high range marker to visualise the integrity of the DNA. To check quantity we strongly recommend using an intercalating dye/fluorometric quantification and actively discourage use of spectrophotometric methods. DNA which appears to ‘smear’ down the gel is indicative of degraded genomic DNA, whereas DNA which generates a tight band is indicative of intact DNA.

We recommend DNA be extracted using methods that yield higher quality DNA, such as column, bead based, or C-TAB extraction. We would discourage use of "crude" DNA extractions.

We do not recommend using a total DNA measurement system (e.g. Nanodrop) as this will not differentiate between dsDNA and other contaminants which could affect the result. We recommend using Qubit™ or PicoGreen™ systems for DNA concentration measurement.

For Flex-Seq the standard targeted depth per locus is a range between 75-100x. This depth is the average for the entire panel, which means some loci will receive higher or lower depth. Accurate SNP calling is routinely possible with 10x coverage per haploid copy of the genome.

We do not recommend using deep well plates, as they may lead to higher moisture content and degradation of the samples. We would recommend using the BioArk Leaf Collection Kit or BioArk Seed Collection Kit.

For a list of plant species with an optimised extraction protocol available, please contact technical and applications support or email technical and applications support for the most up to date information.

Yes, on the condition that the DNA quality meets minimum requirements and as long as reference genome and SNP information/locations have been identified. All Flex-Seq projects require:

• a reference sequence (genomic or transcriptomic, or a reference scaffold). These can be provided as a link to a publicly available genome or submitted as a FASTA file.
• a list of SNP locations to be interrogated, submitted as a VCF, Excel or BED file.

Please contact technical and applications support for further information if you have any queries about your organism.

DNA quality and concentration are critical to the success of Flex-Seq, and typically, DNA from FFPE samples is of suboptimal quality. We have experience working with FFPE samples, and so please contact technical and applications support for further advice before proceeding.

Yes, there is no reason why a WGA method cannot be used to increase the DNA concentration of your sample. Please be aware that any polymerase based amplification may introduce an element of bias and/or PCR errors within your target. Also, the DNA will need a post-amplification clean-up step prior to submission.

For Flex-Seq projects, we only accept DNA samples in 96-well plate format.

Flex-Seq relies on a specific DNA starting concentrations, quality, and volume. Should we still have the primary samples/DNA in our possession, these will need to be re-evaluated to determine if the samples meet the minimum requirements. However, all-inclusive KASP projects do not use RNAse, which is highly recommend for the Flex-Seq protocol, which may impact the results. Please contact technical and applications support for further information.

We do not recommend lowering the DNA amount dramatically, as the library preparation has been validated to require 20 µL at 30 ng/µL. In case that the DNA is highly purified and > 10kb it is possible to submit concentrations of approximately 20 ng/µL but the volume should be no less than 20 µL. Please contact technical and applications support for further information.

Initial library design and set-up takes approximately 6 - 8 weeks. Re-orders of the same Flex-Seq assay design take about 3 weeks. Once the Flex-Seq library is available, the expected turnaround time including bioinformatic analyses is 3 weeks from receipt of samples. Please contact the Flex-Seq team for a detailed turnaround time breakdown and up-to-date pricing information.

No, this is not possible as they are different technologies. KASP works by directly targeting the SNP, whereas Flex-Seq probes are designed to the surrounding sequence to the SNP of interest. Using Flex-Seq may only be possible if the locations of the SNPs in the KASP assay can be mapped back to a specific location in a reference sequence.

One of the requirements for a Flex-Seq service is the genome location (not necessarily the make-up) of each SNP to be interrogated. Unfortunately, the KASP panel assays do not have this location information in their panel database, as this is not required for KASP assay design/ordering, but the KASP panel may be used as a starting point for the Flex-Seq panel design.

The QC of the submitted DNA prior to library preparation is part of the Flex-Seq service. However we recommend DNA be extracted using methods that yield higher quality DNA, such as column, bead based, or C-TAB extraction to minimise factors such as secondary compounds/metabolites, chemical carryover from extraction, or excessive degradation. We discourage use of "crude" DNA extractions.

We do not recommend using deep well plates, as they may lead to higher moisture content and degradation of the samples. We would recommend using the BioArk Leaf Collection Kit or BioArk Seed Collection Kit.

Yes, on the condition that the DNA quality meets minimum requirements and as long as reference genome and SNP information/locations have been identified. All Flex-Seq projects require:

• a reference sequence (genomic or transcriptomic, or a reference scaffold). These can be provided as a link to a publically available genome, or submitted as a FASTA file.
• a list of SNP locations to be interrogated, submitted as a VCF, Excel or BED file.

Please contact technical and applications support for further information if you have any queries about your organism.

DNA quality and concentration are critical to the success of Flex-Seq, and typically, DNA from FFPE samples is of suboptimal quality. We have experience working with FFPE samples, and so please contact technical and applications support for further advice before proceeding.