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Data from: Applying PCR cycle autonormalization to PacBio full-length 16S rRNA library preparations: impacts on error rates and sequence distributions

Published by Agricultural Research Service | Department of Agriculture | Catalog Last Checked: August 03, 2026 at 05:01 PM | Dataset Last Updated: July 10, 2026
The bacterial 16S rRNA gene is used extensively for the study of host and environment microbiomes. These approaches have most often relied on sequencing partial fragments of the 16S (250-400bp). Improvements to PacBio sequencing in both quality and yield may provide additional sequence resolution while improving sampling depth. Despite this, PCR-based errors remain an outstanding issue, and this could be further compounded with these techniques due to a five-fold increase in sequence coverage. Here, we addressed the issue of overamplification of PCR and evaluated how seven different specimen types performed under four different cycling procedures. We compared conventional PCR at different cycle thresholds (20, 24, 30) to PCRs that were automatically terminated on a per-sample basis by amplification thresholds (autonormalization) on the iconPCR instrument. Overall, our results demonstrate that autonormalization protocols improve PacBio Kinnex sequencing by reducing PCR introduced errors in over cycling, while ensuring all samples receive a necessary amount of amplification. This dataset is in support of these underlying conclusions.

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Complete Metadata
@type dcat:Dataset
accessLevel public
bureauCode 
[
  "005:18"
]
contactPoint 
{
  "fn": "Mason, Charles J.",
  "hasEmail": "mailto:charles.mason@usda.gov"
}
description <p dir="ltr">The bacterial 16S rRNA gene is used extensively for the study of host and environment microbiomes. These approaches have most often relied on sequencing partial fragments of the 16S (250-400bp). Improvements to PacBio sequencing in both quality and yield may provide additional sequence resolution while improving sampling depth. Despite this, PCR-based errors remain an outstanding issue, and this could be further compounded with these techniques due to a five-fold increase in sequence coverage. Here, we addressed the issue of overamplification of PCR and evaluated how seven different specimen types performed under four different cycling procedures. We compared conventional PCR at different cycle thresholds (20, 24, 30) to PCRs that were automatically terminated on a per-sample basis by amplification thresholds (autonormalization) on the iconPCR instrument. Overall, our results demonstrate that autonormalization protocols improve PacBio Kinnex sequencing by reducing PCR introduced errors in over cycling, while ensuring all samples receive a necessary amount of amplification. This dataset is in support of these underlying conclusions. </p>
distribution 
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identifier 10.15482/USDA.ADC/30964789.v1
keyword 
[
  "Agriculture",
  "Arthropod",
  "bacteria",
  "environmental",
  "metabarcoding",
  "metagenomic",
  "microbiome",
  "soil",
  "source code"
]
license https://creativecommons.org/publicdomain/zero/1.0/
modified 2026-07-10
programCode 
[
  "005:040"
]
publisher 
{
  "name": "Agricultural Research Service",
  "@type": "org:Organization"
}
temporal 2025-07-01/2025-07-01
title Data from: Applying PCR cycle autonormalization to PacBio full-length 16S rRNA library preparations: impacts on error rates and sequence distributions

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