What stops clusters from merging during bridge amplification?
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3.7 years ago
27atcggcta27 ▴ 180

Hello Everyone!

I'm sorry if this is answered somewhere else, If it is please direct me to it. I cant seem to find much information.

I'm wondering how DNA fragment clusters dont get all mushed together on a flow cell when sequencing after bridge amplification.

It is my understanding that during RNA-Seq, RNA is converted to DNA, then the DNA is fragmented. The resulting fragments are layed out on a flow cell, amplified by bridge amplification to get stronger light detection, and then sequenced.

So my confusion stems from this thought: If DNA fragments are dispersed randomly throughout a flow cell, and then amplified by bridge amplification, what is preventing the clusters from merging? I made a picture (see link below) to better describe my question.

Pictorial depiction of my confusion

I guess my question could also be reworded as "What prevents DNA fragments from amplifying too close to each other in a flow cell?"

Thank you in advance!

RNA-Seq sequence • 804 views
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3.7 years ago
GenoMax 143k

This is not a bioinformatics question but briefly

What prevents DNA fragments from amplifying too close to each other in a flow cell?

For a patterned flowcell, the physical presence of a nano-well, which is a defined physical well.

For non-patterned flow cells nothing prevents that. Clusters can indeed touch or merge/mix. "Chastity filtering" included in bcl2fastq pre-processing detects and removes any clusters that fail this cluster purity filter.

Accurate quantitation of libraries and appropriate loading concentrations are keys to minimizing this problem.

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Excellent, Thank you very much. This answers my non-bioinformatics question

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