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How are distances between clusters in Illumina sequencing (resp. bridge PCR) achieved?

In Illumina sequencing by synthesis are DNA fragments amplified by bridge PCR. Each fragment gets an adapter which is complementary to adapters on flow cell surface. ssDNA fragments bind to adapters on flow cell and then PCR begins and from each fragment is created cluster of clones. But what if two different initial fragments are binded so close that created clusters are also so close and consequently in sequencing, base calls from each cluster cannot be distinguished?

sequencing illumina
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updated by Devon Ryan Devon Ryan • written by jirkanov • jirkanov

base calls from each cluster cannot be distinguished

How so? Cluster(s) consistently generating discordant base calls will be filtered out by chastity filter in Illumina software (see the explanation of chastity filter in this note).

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written by GenoMax GenoMax

1 answer

What you're describing happens all of the time and the clusters are flagged before fastq files are ever made. For the most part, the base call quality on such clusters is very very low and even if the cluster somehow passes filter by Illumina's software, it's unlikely to survive after trimming or actually get mapped (if one skips trimming).

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written by Devon Ryan Devon Ryan

Thanks. I am just curious: how many templates are lost by this?

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written by jirkanov • jirkanov

You will see a percent clusters passing filters (PF%) stat on Illumina demultiplexing reports. Some fraction of clusters that fail the filter are attributable to the effect you described. Though I don't think there is an easy way of determining the exact number.

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written by GenoMax GenoMax

It varies by the cluster density: the more clusters you have, the more likely that collisions/overlaps occur.

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written by harold.smith.tarheel harold.smith.tarheel

To toss some numbers, my last run on a MiSeq had a PF% of 94%, from which 96% had a quality score of >30.

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written by ATpoint ATpoint

To keep things in perspective (since numbers have been mentioned) with patterned flow cells a PF% of ~75% is considered optimal.

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written by GenoMax GenoMax

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