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The remaining read that was uncovered in the Dicer KO library had a 5' end that did not map to the 5' or 3' end of the hairpin suggesting that it was a degradation product of the full length hairpin.
The remaining read (GCFF90V2H8EV7), which showed higher identity with the nuclear genome, may represent a more ancient insertion.
Remaining read pairs and orphans were assembled with the Trinity de novo assembler [ 71] in paired-end mode.
Using the SSAHA pileup pipeline (ftp://ftp.sanger.ac.uk/pub/zn1/ssaha_pileup/), single nucleotide polymorphisms (SNPs) and short indels (1 3 bp) are called from the remaining read pairs.
Although the length of each remaining read sequence is 50 nt, four nucleotides from the 3′-terminus were excluded for accuracy, following the SOLiD whole-transcriptome analysis protocol.
Next, we took 25nt of 5′- as well as 3′-end of each remaining read (∼17.8% of total reads) and used these for further analyses.
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Remaining reads were mapped to P. putida KT2440 genome using Bowtie with the default parameters.
Next, the low-quality reads were filtered, and the remaining reads were used for the additional mapping steps.
The remaining reads were used to predict novel miRNA candidates using the updated miRanalyzer webserver (Hackenberg et al., 2011).
Low-quality reads (with an average quality value below 8) were filtered and the remaining reads were used for the additional mapping steps.
The remaining reads were discarded.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com