This resource provides step-by-step guidance through the process of transitioning to AmpliSeq for Illumina on the iSeq 100 Sequencing System.
The total library prep time for AmpliSeq for Illumina is 5–7 hours. The hands-on time is < 1.5 hours, not including library quantification, normalization, and pooling.
The AmpliSeq for Illumina workflow does not require on-site training. If you are interested in training, contact your local sales representative for information about the available courses.
The following online video courses are available:
AmpliSeq for Illumina is compatible with samples where available input quantity and quality are not limiting, such as blood, cell culture, or fresh-frozen tissues as well as challenging sample types, such as formalin-fixed paraffin-embedded (FFPE) tissue. The input requirement for DNA and RNA ranges from 1 ng to 100 ng, depending on the application needs. For most applications, we recommend 10 ng input per pool.
DNA purity must have an A260/A280 ratio of 1.8–2.0. PicoGreen (DNA) or Qubit DNA HS kits are recommended for accurate quantification. See the reference guide for your AmpliSeq panel to determine any modifications required.
AmpliSeq for Illumina requires an AmpliSeq for Illumina panel, AmpliSeq for Illumina Library PLUS Kit, and compatible AmpliSeq index kits. The AmpliSeq for Illumina Library PLUS Kit is available in 24-, 96-, and 384-reaction configurations.
For information on index kit compatibility, refer to the Reference Guide for your panel.
For information on the required equipment and user-supplied consumables, visit the support page for your panel. A consumables and equipment list can be found on the Documentation page for each panel.
AmpliSeq for Illumina libraries are manually normalized before pooling.
The library is automatically denatured into single strands and further diluted onboard the instrument. For more information, see the clustering information and Library Dilution section in the iSeq 100 Sequencing System Guide.
Indexing is a way to label biological samples, then use bioinformatic methods to distinguish the sequencing data generated during a run.
For more information, see the Dual-Indexed Workflow on a Paired-End Flow Cell (Workflow B) section of the Indexed Sequencing Overview Guide.
1–96 samples can be processed at a time through the protocol. However, it is recommended to process 8 samples or multiples of 8 using a multichannel pipette.
The index adapters used in AmpliSeq for Illumina protocols are specifically designed for AmpliSeq for Illumina. Adapters have proprietary design and modifications. Nextera or TruSeq adapters are not compatible with this protocol.
See the AmpliSeq for Illumina Panels section of the Illumina Adapter Sequences Document for the adapter sequences and the AmpliSeq for Illumina Pooling Guidelines section of the Index Adapters Pooling Guide.
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 reference guides.
For recommendations on thermal cyclers, visit the support page for your AmpliSeq for Illumina panel. A consumables and equipment list can be found on the Documentation page for each panel.
For more information, view the Sequencing: Illumina Technology video or refer to An Introduction to Next-Generation Sequencing Technology.
The iSeq 100 System requires the iSeq 100 i1 Reagents kit. For information on required user-supplied consumables and equipment, see the iSeq 100 Sequencing System Guide.
Refer to the Set Up a Sequencing Run (Local Run Manager Mode) or Set Up a Sequencing Run (Manual Mode) sections of the iSeq 100 Sequencing System Guide.
You can use the iSeq 100 System Sample Sheet Template to create the sample sheet. A sample sheet is required when sequencing in Manual mode with BaseSpace Sequence Hub Run Monitoring and Storage. Download and edit the sample sheet, then upload it to the control software during run setup.
The optimal sequencing depth varies depending on the application you are running and your experimental goals. Refer to the literature for applicable reference studies.
For the length and application of the panel, 2x151 bp is recommended.
Refer to the Prepare Libraries for Sequencing and the Cluster Generation sections in the iSeq 100 Sequencing System Guide.
See the technical bulletin What is the PhiX Control v3 Library and what is its function in Illumina NGS?
Refer to the Number of Cycles in a Read section in the iSeq 100 Sequencing System Guide.
Paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.
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 (NGS) systems are capable of paired-end sequencing. Illumina recommends paired-end sequencing for AmpliSeq for Illumina libraries.
Patterned flow cells contain billions of nanowells at fixed locations across both surfaces of the flow cell. The structured organization provides even spacing of sequencing clusters to deliver significant advantages over non-patterned cluster generation.
Refer to the Introduction to Key Concepts in Sequencing Data Analysis webinar for an introductory presentation and discussion on the concepts and general approaches used in analyzing Illumina sequencing data. This webinar is targeted to new to Illumina NGS users and covers the basic analysis approaches used in de novo assembly, RNA sequencing, and SNP finding. The webinar also describes basic concepts in experimental design.
Local Run Manager is a Windows-based software for Illumina benchtop sequencing data, which 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.
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.
Refer to the following resources:
The DNA Amplicon and RNA Amplicon applications are both available through BaseSpace Sequence Hub.