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This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos.
However, less easily detectable sequence errors can be introduced by another intrinsic property of RTs.
Rigorous quality measures are applied to separate PCR amplification and/or sequence errors from true polymorphisms.
This number is generally a lower limit, as various biases and sequence errors often reduce the quality of sequence reads.
A sequence quality control excluded puteins with gaps and sequence errors that caused five or more undetermined amino acids.
More typically, clones not resulting in detectable GFP expression had minor sequence errors including amino acid substitutions.
To minimize sequence errors during PCR amplifications, a high fidelity Phusion DNA polymerase (New England Biolabs) was used for all PCR amplifications.
Once we eliminated presumed N2 reference sequence errors, we identified 1208 differences between N2 and LSJ1, comprising 877 SNPs and 331 indels (Table S2).
Sequence analysis of the final fusion product was used to verify lack of sequence errors and that the hfq ORF was replaced with the ermC ORF.
The sequence errors observed are therefore not reflective of DNA diagenesis in the CPC98 but are most likely artefacts of the sequencing method used.
We used PHRED score 15 as cutoff value for quality sequences because the most meaningful annotation analysis is BLASTX that has high tolerance to minor sequence errors.
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