Question: How are distances between clusters in Illumina sequencing (resp. bridge PCR) achieved?
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gravatar for jirkanov
3.0 years ago by
jirkanov10
jirkanov10 wrote:

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 • 1.2k views
ADD COMMENTlink modified 3.0 years ago by Devon Ryan95k • written 3.0 years ago by jirkanov10

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).

ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by genomax83k
2
gravatar for Devon Ryan
3.0 years ago by
Devon Ryan95k
Freiburg, Germany
Devon Ryan95k wrote:

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).

ADD COMMENTlink written 3.0 years ago by Devon Ryan95k

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

ADD REPLYlink written 3.0 years ago by jirkanov10
2

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.

ADD REPLYlink written 3.0 years ago by genomax83k
1

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

ADD REPLYlink written 3.0 years ago by harold.smith.tarheel4.5k
1

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

ADD REPLYlink written 3.0 years ago by ATpoint34k

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

ADD REPLYlink written 3.0 years ago by genomax83k
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