Reagents in Rapid SR cluster and Rapid SBS kit

[biancaneve]

Hi I am a new user of the hiseq sequencer. I know the workflow of cluster generation and sequence by synthesis. I am interested in knowing how each of the reagents provided in Rapid SR cluster kit and Rapid SBS kit are involved in these 2 processes.

Hope someone can help me with it as I cannot find any information on it or is there any places i can find the resources? Thanks!

[matth431]

Originally Posted by biancaneve

Hi I am a new user of the hiseq sequencer. I know the workflow of cluster generation and sequence by synthesis. I am interested in knowing how each of the reagents provided in Rapid SR cluster kit and Rapid SBS kit are involved in these 2 processes.

Hope someone can help me with it as I cannot find any information on it or is there any places i can find the resources? Thanks!

The rapid cluster kit provides all of the reagents needed to cluster the library onboard the HiSeq2500 machine - the diluted library goes into the FCA or FCB template position to the left of the flowcell stage (the two Eppendorf tube positions). The cluster kit reagents sit in the same position in the machine as the paired-end reagents for a standard high-output run (and, if doing a rapid PE run, also contain tubes of the PE turnaround reagents, together with the read 1/2 and i7/i5 sequencing primers).

The rapid SBS kit is much the same as the standard high-output SBS reagents (fluorescent dNTP and polymerase mix, scan reagent, salt buffers and cleavage mix), but there are some reagents used in high-output which are not needed for the rapid run and are thus replaced with dummy PW1 corning tubes. Unlike high-output, the sequencing mix for the rapid run comes with the polymerase and flourescent dNTPs already spiked in.

If you don't want to cluster onboard the instrument (since there's only one template position for each flowcell, both lanes of that flowcell must be loaded with the same library... so if you want lane 1 and lane 2 to have different samples loaded, you cannot cluster onboard) you need a third kit, the cBot Rapid Duo kit. This allows loading of DNA onto the flowcell via a cBot instrument, but only does the first round of the clustering (takes about 1hr 20min) - the rest is done onboard the HiSeq using the standard Rapid Cluster Kit.

Note that flowcells dual-loaded with the cBot are NOT stable for 9 days prior to running (as they are with regular cBot-loaded high-output cells) since the clustering is not yet complete... they have to be loaded onto the HiSeq on the same day.