Differential amplicon size effects on clustering across Illumina instruments

pmiguel

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Join Date: Aug 2008

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Differential amplicon size effects on clustering across Illumina instruments

04-18-2019, 08:35 AM

I would like to recommend to your attention a Bioarchive (pre-print) paper:

Measuring Illumina Size Bias Using REcount: A Novel Method for Highly Accurate Quantification of Engineered Genetic Constructs

https://doi.org/10.1101/388108

Quantification of DNA sequence tags associated with engineered genetic constructs underlies many genomics measurements. Typically, such measurements are done using PCR to enrich sequence tags and add adapters, followed by next-generation sequencing (NGS). However, PCR amplification can introduce significant quantitative error into these measurements. Here we describe REcount, a novel PCR-free direct counting method for NGS-based quantification of engineered genetic constructs. By comparing measurements of defined plasmid pools to droplet digital PCR data, we demonstrate that this method is highly accurate and reproducible. We further demonstrate that the REcount approach is amenable to multiplexing through the use of orthogonal restriction enzymes. Finally, we use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencing platforms.

entitled:
Measuring Illumina Size Bias Using REcount: A Novel Method for Highly Accurate Quantification of Engineered Genetic Constructs

See panel D below:

They add the same molar amount of a bunch off different size constructs and then measure the relative amount of clustering they get.

It looks like the NovaSeq is far less sensitive to amplicon size with regards to clustering efficiency! It only looks like ~20x drop in efficiency going from 150bp to 1500bp!! Whereas for a MiSeq it would be about 5000x drop in efficiency of clustering.

If you are a NovaSeq hater, feel free to hypothesize that this effect is largely the result of long amplicons being able to escape into adjacent (or even cross-surface) wells. Thus you can attribute the NovaSeq advantage to duplicate amplicon clusters...

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Phillip
Tags: cluster density
SNPsaurus

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Join Date: May 2013

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#2

04-18-2019, 09:32 AM

Interesting difference between NextSeq Rapid and HO. The rapid groups with the examp platforms, while HO looks like the other random cluster platforms.

Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com
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Differential amplicon size effects on clustering across Illumina instruments

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