No, a new version is not currently planned. The off-the-shelf panels and Community panels are based on hg19. Conversion of pre-designed panels may be considered in the future based on market demand.
Amplicon can have a range where you select the maximum amplicon size.
Because we digest the primers during the Partially Digest Amplicons step, the resulting insert sizes will be smaller than the amplicon size. Depending on read length chosen, we recommend adapter trimming.
Optimal probes are chosen using an algorithm that considers melting temperature (Tm), % GC, length, secondary structure, uniqueness in the genome, and the presence of underlying SNPs (based on dbSNP). For more information, see the DesignStudio online help.
No. This is a multiplex PCR which will not be able to discern between
the top and bottom strand.
No. However, you can use the “Modify Design” button to copy the content to a new panel and then edit it.
No. After you’ve placed an order, you cannot edit the design. The files necessary for analysis by BaseSpace Sequence Hub and Local Run Manager must remain in sync with the material you ordered.
A design smaller than 250 kb has an expected turnaround time of 48 hours or less. Designs greater than 250 kb or with many targets can take longer than 48 hours to return.
On-Demand designs have a shorter turnaround time than other submissions. On-Demand designs of 250 kb or less should be returned in less than 2 hours.
Currently, DesignStudio allows users to choose an amplicon size of 140, 175, 275, or 375 (recommended for MiSeq) for each design. The amplicon size includes the primer sequences and the insert regions. We recommend using 175 bp for FFPE DNA, 140 bp for cfDNA, and 275 bp for normal DNA.
Yes, DesignStudio enables SNP genotyping by sequencing.
You can submit designs of up to 500 kb directly to the pipeline. The pipeline is capable of processing designs up to 5 Mb, but such designs are costly and take up a large amount of computational resources.
We recommend that you only submit designs up to 2 Mb. For designs between 2 Mb and 5 Mb, we recommend that you contact your sales specialist.
Primers in the same pool/tube do not overlap.
The process is an automated pipeline, optimized to provide the maximum coverage with reliable primer sets.
No. You must modify the design, add the new genes, and submit a new order.
No, not at this time.
No. We use specially modified primers, so standard primers will not allow for library construction.
AmpliSeq for Illumina custom panels range from 12 amplicons to 3,072 amplicons per pool. Target regions can be as small as 1 bp, but because designs must include 12 amplicons, you would need 12 sets of 1 bp regions.
All orders have a minimum price equivalent to the cost of an order containing 48 amplicons.
Each custom primer pool is delivered as a pre-pooled tube.
Email us at email@example.com. Use your AmpliSeq for Illumina Design ID number or Solution ID number when referring to your order.
If your design results in multiple pools, each pool is processed independently through “Amplify DNA/cDNA Targets” as referenced in the AmpliSeq for Illumina Custom and Community Panel Reference Guide. The pools then are combined prior to the “Partially Digest Amplicons” step. They continue as one sample through index ligation and final library amplification.
To make sure that an entire exon is covered, by default, we add 25 bp of padding up and down-stream of the selected target region. This padding allows for room to place the primers. Padding ensures high-quality sequencing at the ends of the exons and allows some sequencing into the splice junction regions. Primer regions are not considered covered. Therefore, if coverage obtained from the initial design is less than 100%, we can try one more time to extend the primer further into the intron to capture the whole exon.
The assay uses between 1 and 100ng of DNA per primer pool, with most designs using 10ng per pool.
We’ve seen success with low quality inputs using the protocol modifications indicated in the user guides. Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8–2.0. PicoGreen is recommended for an accurate quantification.
Only use FFPE-derived DNA when using short amplicon lengths of 140 or 175 bp. Shorter amplicons provide better amplification than longer ones when the sample input is fragmented FFPE-derived DNA.
There is a limit of 12-6,144 primer pairs per pool. If generating target region greater than 5Mb, we recommend selecting an enrichment option.
The adapters used in this assay are optimized for the AmpliSeq workflow. Nextera or TruSeq Adapters are not compatible with this assay.
See Contents & Storage.
It is possible to run 3 different AmpliSeq for Illumina designs each with barcodes on the same sequencing run. However, your target amplicon size and required coverage must be achieved in a single run
A 2×150 bp paired-end read is recommended for 140-275 bp amplicon sizes. Up to 2x300 bp paired-end run on the MiSeq is recommended for 375 bp amplicon sizes.
This kit has integrated sample barcodes that enable pooling of up to 96 samples per sequencing run. However, the actual number of samples that can be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. An online calculator is provided in DesignStudio to help with these calculations.
Local Run Manager and BaseSpace Sequence Hub have apps available for analysis. The DNA Amplicon Analysis App and RNA Amplicon Analysis App are available on BaseSpace Sequence Hub. Further
analysis can be performed on any variant calls using BaseSpace Variant Interpreter. Local Run Manager has a similar DNA Amplicon Analysis Module and RNA Amplicon Analysis Module which utilizes the same workflow and algorithm as the BaseSpace Sequence Hub Apps. The DNA Amplicon analysis workflow can be used to perform alignment and variant calling and the RNA Amplicon analysis workflow for fusion calling or differential expression analysis. Additionally, OncoCNV caller, a BaseSpace Lab Apps is available for CNV analysis.
For an example data from an experiment, go the Public Data page in BaseSpace.
DesignStudio returns high confidence amplicon designs that have delivered unprecedented amplicon multiplexing performance. Since each design is unique and sample input can vary, performance of the design will need to be tested empirically.
We’ve set an ordering minimum of 1 gene or 24 amplicons per panel. Designs must also have at least 2 pools and 12 amplicons per pool.
We have set an ordering maximum of 500 genes or 15,000 amplicons per panel due to manufacturing restrictions. We are always making improvements, so this limit is likely to increase. You may be able to order larger designs in the future.
Illumina uses RefGene v74 as the source of annotations.
No, only the coding DNA sequence (CDS) region of a gene is included as part of an On-Demand gene design.
Gene amplicon uniformity is the percentage of amplicons for a gene with greater than 0.2 times the mean coverage of all amplicons targeting that gene. It represents the observed wet-lab uniformity calculated from NextSeq data with the Illumina DNA Amplicon workflow.
No. On-Demand panels only support genes containing CDS regions. Pseudogenes are not supported.
The padding for every On-Demand gene design is 5 bp on the 5′ and 3′ ends of the exon.
No. The number of possible combinations is astronomical. It is not feasible to test for all possible combinations in the lab. However, through computer-based searches, we have reduced the occurrence of primer-primer interactions as much as possible. In addition, when synthesizing many genes simultaneously in large batches, we have observed less than 1% amplicon drop-out due to suspected primer-primer interactions.
The number of amplicons per pool in DesignStudio reflects the number of unique amplicons in each pool. The number of primer pairs per pool on the tube and box labels reflects the total number of oligos per pool. Either value can be used when preparing libraries according to the AmpliSeq for Illumina On-Demand, Custom and Community Panels Reference Guide (Table 4. X cycles and X minutes). If the values fall into different cycle categories, the higher PCR cycle number is recommended.
The “observed coverage” track indicates the number of observed reads for each amplicon of each targeted gene during validation experiments on a NextSeq. Use this track as general guidance for the likely performance when running an experiment. While values can vary among assays, the general coverage trend should remain consistent.
Gaps occur where there are no amplicons to provide coverage for the intended target. We have made every effort to minimize the occurrence of these regions in our On-Demand designs.
The Y-axis represents the observed coverage normalized by the mean amplicon coverage for the gene.
No. The IGV viewer can only focus on your gene of interest. In the Grid View, select a gene, and the IGV viewer updates automatically to center on that gene.
All amplicons in the design contain reads that are visualized in the “observed coverage” track. If the number of reads covering an amplicon is relatively small in comparison to neighboring amplicons, the “observed coverage” track appears empty. However, if you change the scale to a lower value, you will then be able to visualize the lower number of reads. If the observed coverage track is not present, the designer notifies you why that track is not available.