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The assembly was conducted using the MIRA assembler program version 3.1.15 [ 36] with default settings and the 454 reads from the normalized, the non-normalized and the combined libraries.
Genome assembly was conducted de novo with ABySS assembler using paired end libraries (Simpson et al. 2009).
The assembly was conducted with the aim of co-assembling ESTs of orthologous genes (including homoeologous pairs of genes in B. napus from each of the A and C genomes), but resolving assemblies of paralogous, or paleo-homoeologous, genes (i.e. the genes related by the ancestral genome triplication observed in diploid Brassica species).
Due to the heuristic nature of the assembly process and previous reports of redundancy (different contig sequences belonging to the same transcript region) in sets of transcriptome contigs assembled with different methods [ 17], a second run of assembly was conducted using the previously obtained contigs and singlets as input.
Transcriptome assembly was conducted using Trinity, a de Bruijn graph-based de novo assembler that has shown high performance in recovering full-length transcripts and splice isoforms [ 12, 46, 47].
De novo assembly was conducted using SPAdes (Bankevich et al. 2012), Velvet (Zerbino and Birney 2008), and IDBA-UD (Peng et al. 2012), respectively.
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Modal analyses of an integrated assembly were conducted by employing the fluid-structure (F-S) model as well as the traditional added-mass model.
Base calling and contig assembly were conducted using Phred/Phrap and visualized with Consed [31], [32], [33].
Sequence management and contig assembly were conducted using Sequencher 4.9 software.
Library preparation, sequencing, and assembly were conducted under contract at GATC Biotech AG (Konstanz, Germany).
Two runs of assembly were conducted by MIRA 3 in "EST" and "accurate" usage mode, respectively.
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