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Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Published on: August 3, 2018

454 antibody sequencing - error characterization and correction.

Ponraj Prabakaran¹, Emily Streaker, Weizao Chen

¹Protein Interactions Group, Center for Cancer Research Nanobiology Program, National Cancer Institute (NCI)-Frederick, National Institutes of Health (NIH), Frederick, MD 21702-1201, USA. dimiter.dimitrov@nih.gov.

BMC Research Notes

|October 14, 2011

Summary

This summary is machine-generated.

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454 sequencing of antibody repertoires shows up to 20% errors, mainly from insertions in homopolymers. Error correction reduces this to 5-10%, but some errors persist, requiring caution for antibody variant discovery.

Area of Science:

Molecular Biology
Immunology
Bioinformatics

Background:

454 sequencing is crucial for analyzing antibody repertoires with millions to trillions of unique molecules.
Identifying and correcting sequencing errors is vital for studying antibody maturation and germline origins.
High-throughput sequencing of antibody repertoires presents challenges in error detection.

Purpose of the Study:

To quantify and correct sequencing errors in 454 sequencing of antibody repertoires.
To assess the impact of sequencing errors on antibody variant identification.
To evaluate the effectiveness of error correction strategies for antibody sequencing data.

Main Methods:

Sequencing of six known antibodies at varying concentrations using 454 technology.

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Comparison of 454 sequencing results with gold-standard Sanger sequencing data.

Analysis of error types, including insertions, deletions, and variants at homopolymer regions.

Main Results:

454 sequencing introduced approximately 20% incorrect reads, primarily due to insertions in short homopolymer regions (2-3 nucleotides).
Error correction methods reduced erroneous reads to 5-10%.
A persistent 4-8% of errors remained uncorrected without repeated sequencing, which is impractical for large antibodyomes.

Conclusions:

454 antibody sequencing exhibits a high error rate (up to 20%) that can be reduced but not eliminated.
Reduced error rates (5-10%) still necessitate careful interpretation to prevent false discoveries of antibody variants.
Caution is advised when analyzing antibody diversity from 454 sequencing data due to residual errors.