Questions & Answers

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General

Will there be a new version of the Ready-to Use and Community AmpliSeq for Illumina research panels based on hg38?

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.

DesignStudio

What are the target amplicon sizes?

Amplicon can have a range where you select the maximum amplicon size. 

Setting

Minimum

Maximum  
140 125 140  
175 125 175  
275 125 275  
375 125 375  

Are amplicon sizes equivalent to insert sizes?

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. 

Why are some targets difficult to design in DesignStudio?

  • Homologs: Having homologs in the same design can lead to low designability. Split homologs into separate pools.
  • GC Content: Regions with greater than 80% GC content can be difficult to design against, particularly when these regions are greater than 500 bp in length.
  • Homopolymer sequences and Repetitive elements: DesignStudio avoids these regions to make sure that probes have better specificity in the genome.
  • Poor Specificity: DesignStudio will assess the specificity of probes and exclude those which will not provide satisfactory on-target coverage.

How does DesignStudio select primers?

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.

How can I improve the designability and coverage in my DesignStudio Project?

  • Increasing the size of the target to design against can rescue previously ‘undesignable’ regions. The increased size of a target gives DesignStudio a little more flexibility to fit a higher scoring amplicon over the desired target bases.
  • Change the context of the panel – for example, putting a highly homologous or high GC rich target sequence into the same multiplex design can be problematic for designing probes to amplify each target discretely. Moving problematic regions into a separate design can frequently improve the designability.
  • Change the stringency levels.

Can I create a dual pool design?

No. This is a multiplex PCR which will not be able to discern between the top and bottom strand.

Can I edit the content of my design after I’ve submitted it?

No. However, you can use the “Modify Design” button to copy the content to a new panel and then edit it.

Can I edit my design after I’ve placed an order?

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.

What is the current turnaround time for a submitted design with respect to the target size or number of targets?

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.

What determines the amplicon design criteria?

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.

Is it possible to use an AmpliSeq for Illumina design to screen many SNPs (up to 1000 or more) for many individuals (up to 1000 or more)?

Yes, DesignStudio enables SNP genotyping by sequencing.

What is the largest design that I can submit to the AmpliSeq for Illumina pipeline?

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.

DesignStudio - Primer Bioinformatics

What is the level of overlap among primers?

Primers in the same pool/tube do not overlap.

With AmpliSeq for Illumina projects in DesignStudio, are primer sets designed automatically (with a computer program), without interrogation from a research scientist?

The process is an automated pipeline, optimized to provide the maximum coverage with reliable primer sets.

DesignStudio - Oligo Ordering

Can I add a few more genes to a set of previously ordered primers?

No. You must modify the design, add the new genes, and submit a new order.

If I have regular primers for a region and I know that they are working, can I add them to my AmpliSeq for Illumina design?

No, not at this time.

Can I add primers manually, postdesign, to cover a region completely?

No. We use specially modified primers, so standard primers will not allow for library construction.

Is there a minimum order for AmpliSeq for Illumina Gene and Hotspot designs?

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.

In what container format should I expect to receive my custom primers?

Each custom primer pool is delivered as a pre-pooled tube.

How can I find out the status of a design submission to AmpliSeq for Illumina Custom Panel?

Email us at customerservice@illumina.com. Use your AmpliSeq for Illumina Design ID number or Solution ID number when referring to your order.

DesignStudio - Troubleshooting and Validating

Suppose that I am targeting a region and DesignStudio suggests a design consisting of two primer pools. For each sample, should I prepare a library for each amplification (each pool)? Or should I combine the two amplifications (the products of the two amplified pools) and then prepare the library?

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.

How many base pairs separate the primers from the target region?

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.

Input

What DNA input amount is required?

The assay uses between 1 and 100ng of DNA per primer pool, with most designs using 10ng per pool.

What quality of DNA is required and how should DNA quality be assessed?

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. 

Are FFPE samples supported?

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. 

How much DNA can be targeted with this kit?

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.

Protocol

Does this assay use the standard Nextera or TruSeq Adapters?

The adapters used in this assay are optimized for the AmpliSeq workflow. Nextera or TruSeq Adapters are not compatible with this assay.

What is required to purchase from Illumina?
Can I perform two or three different amplifications and then pool them before going into library prep?

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

Sequencing

What read length is recommended for sequencing?

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.

How many samples can be sequenced at a time?

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.

Analysis

What tools are offered for data analysis?

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.

What does the data look like? Is there demo data available?

For an example data from an experiment, go the Public Data page in BaseSpace.

What actual assay performance can I expect from my design?

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. 

AmpliSeq for Illumina On-Demand

What is the minimum number of genes I can order in an On-Demand panel?

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.

What is the maximum limit on the number of genes I can order in an On-Demand panel?

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.

What annotation source and version is used to recognize gene symbols when creating an On-Demand Panel?

Illumina uses RefGene v74 as the source of annotations.

Are untranslated regions (UTRs) included in an On-Demand gene’s design?

No, only the coding DNA sequence (CDS) region of a gene is included as part of an On-Demand gene design.

What is “Gene Amplicon Uniformity”?

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.

Do On-Demand panels support UTR-only genes? What about pseudogenes?

No. On-Demand panels only support genes containing CDS regions. Pseudogenes are not supported.

What is the padding used for On-Demand gene designs?

The padding for every On-Demand gene design is 5 bp on the 5′ and 3′ ends of the exon.

Have all possible gene combinations been tested for primer-primer interactions?

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.

Why are the number of primer pairs per pool indicated on the tube and box labels different than the number of amplicons per pool indicated in DesignStudio?

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.

AmpliSeq for Illumina On-Demand – IGV Viewer

What is the “observed coverage” track in the IGV viewer?

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.

What are “Gaps”?

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.

What is the scale on the Y-axis?

The Y-axis represents the observed coverage normalized by the mean amplicon coverage for the gene.

Can I use coordinates to navigate the IGV viewer?

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.

I notice that the “observed coverage” track for an amplicon occasionally does not appear to contain information. Why is that?

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.