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As NAST server limits each submission to 500 pyrotags, an individual library was split into smaller subsets comprising 500 pyrotags, aligned with minimum length set at 200 bp, and with other settings kept at their default values [41].
The assembly resulted in 332,993 transcript contigs with an average length of 1,005 nt and minimum length set to 300 nt.
The presence of putative ORFs in the assembled sequences (with and without the start codon) was determined using Flip 2.0.2, with the minimum length set to 150 amino acids [ 74].
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Similarly, with a minimum length setting for fiber tracts short-distance connections cannot be discovered.
Theoretical open reading frames (ORFs) were first determined using the EMBOSS getorf tool (with minimum ORF length set to 90 nucleotides, and taking all alternative start codons into account).
These C-tails were removed using the "clip adapter sequences script" with the 3' adapter set to CCCCCCCCCCCCCCCCCCCCCCCCCC and the minimum read length set to 26.
De novo assembly of the cleaned read pool was performed using VelvetOptimiser 2.1.7 [ 49] and Velvet 1.0.12 with the minimum contig length set to 100 bp.
Cufflinks program was ran with following parameters, minimum intron length set as 10 nt and a maximum intron length as 10000 nt and assembly was guided by GFF file PlasmoDB-9.3_Pfalciparum3D7.gff PlasmoDB-9.3_Pfalciparum3D7.gff 53].
The sequence parameters (coding content, oligonucleotide composition, and gene length distribution) were estimated in FGENESB for each genome separately through an iterative procedure with the minimum ORF length set to 100 nt.
Adapter removal and quality trimming were performed with the tool AdapterRemoval using the default parameters except the threshold for trimming low quality bases (Q < 10), meaning that consecutive stretches of bases from both the 5' and 3' end of the reads with Phred Quality of 10 or lower were trimmed, and the minimum read length set to 30 nucleotides.
When the minimum length was set to 0.9 kbp, the selected sequences covered more than 96% of the entire SD13M region.
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