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Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.
BMC Genomics. 2011 May 19;12(1):245
Authors: Gilles A, Meglecz E, Pech N, Ferreira S, Malausa T, Martin JF
ABSTRACT: BACKGROUND: The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments. RESULTS: We obtained a mean per base error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but a significant part of the variation is explained by the combination of all seven variables. CONCLUSIONS: The pattern identified here calls for the use of internal controls (e.g. including clones with known DNA sequence) and error-correcting base callers, to correct for errors. For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
PMID: 21592414 [PubMed - as supplied by publisher]
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Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.
BMC Genomics. 2011 May 19;12(1):245
Authors: Gilles A, Meglecz E, Pech N, Ferreira S, Malausa T, Martin JF
ABSTRACT: BACKGROUND: The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments. RESULTS: We obtained a mean per base error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but a significant part of the variation is explained by the combination of all seven variables. CONCLUSIONS: The pattern identified here calls for the use of internal controls (e.g. including clones with known DNA sequence) and error-correcting base callers, to correct for errors. For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
PMID: 21592414 [PubMed - as supplied by publisher]
More...