This resource provides step-by-step guidance through the process of transitioning from Ion Torrent to Illumina next-generation sequencing platforms.
Onsite installation training from Field Application Scientists (FAS) is included with all benchtop system purchases except the iSeq 100 System. These trainings go over experimental design, instrument operation, best practices, and troubleshooting. There are several additional training options available as well.
Ion PGM has accessory equipment that might no longer be needed. Illumina benchtop sequencing consumables are single cartridge-based with a separate flow cell.
The equipment needed for performing an AmpliSeq for Illumina library prep is the same as the equipment needed for performing an AmpliSeq library experiment on the Ion Torrent.
For a list of required consumables and equipment, see AmpliSeq for Illumina Custom and Community Panels Consumables & Equipment.
Sequencing costs vary by system, throughput, and the number of samples per run. Contact your local Territory Account Manager for assistance.
For a comparison of Illumina sequencing systems, see the Sequencing Platform Comparison Tool. Contact Illumina Technical Support or your local Illumina representative for more assistance with making this decision.
All Illumina systems can run AmpliSeq for Illumina. However, due to per-sample output requirements, benchtop systems are typically the most appropriate. For guidance concerning the best fit for your experimental needs, refer to the AmpliSeq for Illumina flyer.
Paired-end sequencing allows users to sequence both ends of a fragment and generate alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts. For more information, see Paired-End vs. Single-Read Sequencing.
Quality scores are measured with Q30 on Illumina systems. Analysis is performed using Local Run Manager or BaseSpace Sequence Hub.
For information on the performance of AmpliSeq for Illumina panels, see the data sheets listed on each of the AmpliSeq for Illumina product pages.
The same sample quality used as input for Thermo Fisher Ion AmpliSeq can be used for AmpliSeq for Illumina.
Overall, the time required for instrument setup and preparation makes the workflow on Illumina systems faster. For more information, see the appropriate product documentation.
| Step | Time |
|---|---|
| AmpliSeq for Illumina library prep | 5–7 hours (can vary with sample number and panel) |
| Sequencing | 17–56 hours (platform and run length dependent) |
| Analysis | 1–2 hours (using Local Run Manager or BaseSpace Sequence Hub) |
The Illumina protocol has a second 7-cycle PCR step at the end of the protocol and an additional round of bead cleanup when compared to the Thermo Fisher Ion AmpliSeq protocol. This difference adds approximately 45–60 minutes total time to AmpliSeq for Illumina library prep (included in above estimates).
Desktop systems use cartridge-based reagents that have different thaw times depending on the system and thaw method. For a full list of thaw and stability information, see How to thaw and store sequencing reagents for optimal performance.
The initial input requirement is the same as the Ion AmpliSeq protocol.
There are several important differences between the Ion AmpliSeq protocol and the AmpliSeq for Illumina protocol, such as added amplification and cleanup steps. To be successful with the AmpliSeq for Illumina protocol, review and follow the instructions in the AmpliSeq for Illumina Custom and Community Panels Reference Guide.
AmpliSeq for Illumina libraries can be quantified using the following:
The manifest format used by Illumina analysis options, such as BaseSpace Sequence Hub or Local Run Manager, is different than the format used in Thermo Fisher Ion Reporter software. Manifests for fixed panels can be obtained from Illumina Technical Support.
The number of samples that can be pooled per run is highly dependent on the panel size and the sequencing system. See the Documentation support page for each panel for recommended sample numbers per instrument.
Because paired-end reads are more likely to align to a reference, the quality of the entire data set improves. All Illumina next-generation sequencing systems are capable of paired-end sequencing.
Ready-to-use panels, Custom panels, Community panels, and On-Demand panels are available and can be ordered through MyIllumina.
After a Custom or Community panel order is placed, the delivery time is within 3–4 weeks.
Talk to your sales representative about how Illumina Concierge can be a resource. Illumina Concierge is similar to Thermo Fisher White Glove Service. Order the appropriate sequencing consumables based on the amplicon size of the panel.
Cluster density is referred to as loading density in the support documentation for the Ion Torrent. For guidance on how to optimize cluster density on Illumina instruments, see the following bulletins:
Although the Ion AmpliSeq custom panels can technically work with the AmpliSeq for Illumina Library PLUS kits, Illumina recommends ordering the custom panels through DesignStudio to achieve optimal support from Illumina.
There are a number of primary quality metrics available during and after sequencing. For guidance regarding which quality metrics are best applied when assessing your run, refer to the training webinar Sequence Analysis Viewer 101: What does it all mean?
The Sequencing Analysis Viewer (SAV) can be installed on or off instrument to quickly view a dashboard of run metrics. More information can be found in the Summary Tab section of the Sequencing Analysis Viewer Software Guide.
Web based trainings are available for benchtop sequencing platforms that explain how to review run quality before further analysis.
Illumina benchtop sequencing platforms run on Windows. You will primarily operate the system through the instrument control software, and the user interface will be familiar to Windows users.
A 2 × 151 bp paired-end run is recommended for 140–275 bp amplicon sizes. Up to 2 × 301 bp paired-end run on the MiSeq system is recommended for 375 bp amplicon sizes.
The kit has integrated sample barcodes that enable pooling of up to 96 samples per sequencing run. 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 available to help with these calculations.
Local Run Manager is a Windows-based software for Illumina benchtop sequencing data. It includes local analysis options, and run and user management. The Local Run Manager webinar provides an overview of Local Run Manager and is targeted at new and intermediate users. For more information, view the Local Run Manager support page and the Local Run Manager Overview training course.
Local Run Manager equipment and computing requirements are available in the Installation section of the Local Run Manager Software Guide.
For information on setting up Local Run Manager, see Sequence File Formats, the FASTQ Processing Tools for Data Analysis webinar, and the Local Run Manager Introduction webinar.
BaseSpace Sequence Hub is the Illumina cloud-based sequencing data analysis solution. For information such as the difference between runs and projects, working with data, and sharing your data, see the BaseSpace Sequence Hub webinar. For additional information, see the BaseSpace Sequence Hub support page and the Preparing Runs with BaseSpace Sequence Hub training course.
As the run progresses, the control software automatically transfers base call files (*.bcl) to the specified output location for data analysis. The method of data analysis depends on your application and system configuration. If you stream your sequencing data to BaseSpace Sequence Hub, it is automatically converted to FASTQ. For more information, see Sequence File Formats.