Implementation Best Practices: Sequencing

For the iSeq 100 System, purchase the iSeq 100 i1 Reagents kit from MyIllumina. The iSeq 100 Sequencing System Guide provides a list of user-supplied consumables and equipment.

For equipment needed to run iSeq, refer to the consumables and equipment section of the iSeq 100 Sequencing System Guide.

Documentation and Literature and Training Videos can be found in the iSeq 100 Sequencing System support page.

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.

The optimal sequencing depth varies depending on the application you are running and your experimental goals.  It is recommended to refer to the literature for applicable reference studies. 

For the length and application of the panel, 2 x 151 bp is recommended.

Refer to the Prepare Libraries for Sequencing and the Cluster Generation sections in the iSeq 100 Sequencing System Guide.

For more information, watch Sequencing: Illumina Technology or refer to An Introduction to Next-Generation Sequencing Technology.

For more information, see the bulletin What is the PhiX Control v3 Library and what is its function in Illumina NGS? or the PhiX Control v3 support page.

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.

Since 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.

Single-read sequencing involves sequencing DNA from only one end, and is the simplest way to use Illumina sequencing. By leveraging proprietary reversible terminator chemistry and a novel polymerase, this solution delivers large volumes of high-quality data, rapidly and economically.

For more information, see the Illumina Website and the Dual-Indexed Workflow on a Paired-End Flow Cell (Workflow B) section of the Indexed Sequencing Overview Guide.

Refer to Optimizing Cluster Density on Illumina Sequencing Systems and Cluster Density Guidelines on Illumina Systems

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.

Clusters can only form in the nanowells, making the flow cells less susceptible to overloading, and more tolerant to a broader range of library densities. Precise nanowell positioning eliminates the need to map cluster sites, and saves hours on each sequencing run. Higher cluster density leads to more usable data per flow cell, driving down the cost per gigabase (Gb) of the sequencing run.

For more information, see the Patterned Flow Cell Technology webpage, video, and technical note.