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The minimum contig size for all assemblies was 200 nucleotides.
We chose 60bp as minimum contig size because conopeptides can be as short as 20 amino acids.
A single reference transcriptome was generated using Trinity package (release: 2013-02-25 with default parameters, kmers-25 and the minimum contig size fixed to 200 bp).
First, low-quality sections of RNA-Seq reads and remaining adaptor sequence were discarded, and the minimum contig size of the Trinity assembly was set to 200 bp.
This system was with a hash length of 31 bp and a minimum contig size of 200 bp, with other parameters set to default values.
Assembly proceeded using the following parameters: word size of 24, bubble size of 50, and minimum contig size of 150 bases.
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The minimum output contig size set to 200 bp with the scaffolding option switched off; all other program settings were left unchanged.
The best assembly (i.e minimum number of contigs with maximum contig size) was obtained with a hash value of 23, resulting in 2,885 contigs with an N50 size of 11,807 bp.
The maximum contig size was 71,434 bp.
Minimum output/retained contig sizes were increased through the assembly using the flags "-lenf 60 1 -lenf 70 5 -lenf 80 20".
The following settings were used: automatic bubble size (50), minimum contig length of 201, automatic word size (23), perform scaffolding, and auto-detect paired distances.
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