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The improved sequences were compared against the original CAF1 sequence using MegaBLAST (Morgulis et al. 2008) with an E-value threshold of 1e-5.
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Improved sequences are submitted to GenBank (2013) and used in studies exploring genome evolution (e.g., see Leung et al., 2010; other manuscripts are currently in preparation).
Improved sequence assemblies were generated for 68 of these BACs (with complete resolution for 29 BACs and reduction of minor misassemblies for 39 BACs).
To identify differences between the CAF1 and improved sequences, the CAF1 sequence was soft-masked using WindowMasker with default parameters.
We also tested whether improved sequence assemblies could be generated by separately altering certain Phrap parameters.
Short truncated compounds (minimal domain), chimerical constructions and improved sequence analogs have been reported.
It should be mentioned here that with the latest much improved sequencing instruments, it would be easier to generate ~ 80 million reads and this would substantially increase the sensitivity of detection in RNA-seq platform.
This improved sequencing capacity can now be used to do genome-wide SNP discovery for non-model organisms [ 9- 12].
The gaps between the contigs were filled using primer walking, and poor quality sequences were improved using specific primers and dye terminator sequencing on automated ABI 3700 and ABI 3730 sequencers (PE-Applied Biosystem).
Five of these sequences were improved in quality by repeating the sequencing procedure.
Multiple alignments of microRNA 169 stem-loop sequences were improved by removing the unreliable regions from the alignment using the web-based program GUIDANCE (http://guidance.tau.ac.il), and NJ phylogenetic tress were created with 2,000 bootstrap replications, and the model/method used was the maximum composite likelihood.
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