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Based upon our previous experimental work [17], we defined a signal as one that has an approximately Gaussian Amplitude peak distribution with a standard deviation of about 7 to 15 bp and a minimum amplitude of at least 700 period-10 VWG/CWB counts (lower horizontal dashed line on the Amplitude axes).
These parameters include a minimum overlap of 40 bp and a minimum overlap identity of 90%.
A minimum overlap of 25 bp and a minimum %ID of overlap of 95%% was used in the secondary assembly.
The assembly parameters were a minimum overlap length of 40 bp and a minimum overlap identity of 95%.
Average length of the contigs was 1125 bp with a maximum of 5302 bp and a minimum of 88 bp.
The assembly used the default kmer size of 25 bp and a minimum contig length of 100 bp.
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We also performed a single assembly of the total reads from all three cDNA libraries, which generated a common transcriptome data set of 36,923 contigs having a minimum length of 300-bp and a minimum mapped read of 50.
A FASTA file of the B. plebeja transcriptome sequence assembly was analyzed in QDD version 1.3 using default parameters: selecting only primers that amplify a PCR product between 90 and 320 bp in length, with a repeat motif of 2 6 bp repeats, and a minimum length of four repeat units.
A threshold alignment score of E-10 was used to filter the results, and only the alignments of at least 100 bp and with a minimum of 95% of identity between the sequences were retained.
We located di-, tri-, and tetra-nucleotide SSRs with lengths less than 50 bp and with a minimum of 4 contiguous repeating units, which provided a large number of candidate SSRs.
The unigenes assembled by short reads from four samples were further clustered into all-unigenes in a comprehensive transcriptome using TGI Clustering Tool TGICLL) with the parameters of 50 bp overlap and a minimum of 90% identity.
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