Lane-by-lane Sequencing Using Illumina's Genome Analyzer II


We came across this cool paper from 2013.

Next-generation sequencing has become an essential tool in molecular biology that has been successfully applied to a broad variety of experimental approaches. While several platforms for next-generation sequencing exist, the most commonly used approach is sequencing-by-synthesis, implemented on Illumina’s Genome Analyzer II (GAII) and HiSeq2000 systems. A key constraint of these sequencers is the need to run multiple lanes of samples with identical parameters as part of a single flowcell. Here, we present a series of modifications to the Illumina Genome Analyzer II, along with a script generating tool, that allow users to run the GAII in a lane-by-lane manner. Any number of lanes can be run at one time. Repeated use of the same flowcell on multiple sequencing runs does not appreciably reduce the intensity, cluster density, or accuracy of the run. These modifications will enable smaller-scale experiments with unusual design parameters to be run routinely on the GAII.

Here is the main method -

Clustering

Lane-by-lane clustering is performed on the Illumina cBot. Clustering kits (Part# GD-3002001 and PE-3002001 for single read and paired-end, respectively, Illumina, Inc., San Diego, CA) were modified by labeling reagent tubes with their reagent number. Tubes corresponding to unused lanes were cut away while frozen (Figure 1a). Unused reagents can be stored frozen for future runs. Empty wells on the reagent plate were replaced with tube strips (Part# MP73004L, Nova Biostorage Plus, Canonsburg, PA) filled with high salt buffer (Solution PR1 in all SBS kits for the Genome Analyzer, Illumina, Inc.) to prevent dehydration of the f lowcell. Lane 1 was always included in the first set of lanes clustered on a given f lowcell to permit edge-finding and tilt- setting.

We have modified Illumina’s GAII to run partial flowcells through minor changes in the instrument and fluidics scripts. These changes are suited for rapid protocol prototyping and sequencing experiments with unusual chemistry requirements.

Imaging

Imaging was limited to desired lanes in order to reduce run time. The XML recipe files were edited to remove the Lane Index line entry for each unused lane under the TileSelection header (Figure 1b). Note that while this does not prevent the brief imaging during calibration, full within-read imaging is restricted to the active lanes. Reagent delivery

In order to preserve high signal-to-noise ratios during longer runs, salt solution in the inactive lanes was removed. Prior to each read, deionized water was flushed across the entire flowcell while the reagent manifold was still in the standard configuration, pre-charging the inactive lanes with salt-free medium.

Following the water wash, reagent flow was physically limited to the desired lanes by inactivating the pumps corresponding to the unused lanes. The plunger ends of the syringe-pumps for the unused lanes were disconnected from the motorized crossbar by removing the connecting screws while in the top position (Figure 1c).

The reagent delivery volume settings were changed to compensate for the effect of fewer pumps on rates of flow.


Written by M. //

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