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Open Access Highly Accessed Technical Note

Assessment of replicate bias in 454 pyrosequencing and a multi-purpose read-filtering tool

Jérôme Mariette*, Céline Noirot and Christophe Klopp

Author Affiliations

Plate-forme bio-informatique Genotoul, INRA, Biométrie et Intelligence Artificielle/Génétique Cellulaire, BP 52627, 31326 Castanet-Tolosan Cedex, France

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BMC Research Notes 2011, 4:149  doi:10.1186/1756-0500-4-149

Published: 26 May 2011



Roche 454 pyrosequencing platform is often considered the most versatile of the Next Generation Sequencing technology platforms, permitting the sequencing of large genomes, the analysis of variations or the study of transcriptomes. A recent reported bias leads to the production of multiple reads for a unique DNA fragment in a random manner within a run. This bias has a direct impact on the quality of the measurement of the representation of the fragments using the reads. Other cleaning steps are usually performed on the reads before assembly or alignment.


PyroCleaner is a software module intended to clean 454 pyrosequencing reads in order to ease the assembly process. This program is a free software and is distributed under the terms of the GNU General Public License as published by the Free Software Foundation. It implements several filters using criteria such as read duplication, length, complexity, base-pair quality and number of undetermined bases. It also permits to clean flowgram files (.sff) of paired-end sequences generating on one hand validated paired-ends file and the other hand single read file.


Read cleaning has always been an important step in sequence analysis. The pyrocleaner python module is a Swiss knife dedicated to 454 reads cleaning. It includes commonly used filters as well as specialised ones such as duplicated read removal and paired-end read verification.