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The designed capture bait pool covered 95.2% of region of interest bases (Supplementary Table 4).
This weakness of the tested enrichment design would also apply to other hybridization-based sequence capture bait designs that subject targets to repeat masking before probe design.
To minimize representational bias of highly conserved regions of the plastid genome (e.g., rRNA genes) during hybridization capture, bait sequences for all genomes were compared using BLAST, and only baits with <90% identity to all other baits were retained in the final bait design.
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BN designed the sequence capture baits.
Using the Agilent target enrichment system, capture baits were designed for GPCRs off the human and rhesus exonic sequence.
It is worthwhile to note that TruSeq and Nextera, both supplied by Illumina, use the same capture baits.
For this reason, longer capture baits and iterative refinement of the bait design would be required for such genomic regions with low complexity.
Bovine Hybloc (Applied Genetics Laboratories, Florida, USA) was used instead of human Cot1 DNA during the sequence capture process to prevent non-specific hybridisation to the sequence capture baits.
As sequence capture baits have some tolerance for mismatches, it is likely that a bait designed for a given gene will capture paralagous genes in cases where sequence divergence is not high.
In order to try and increase the percentage of target capture, we adopted an alternative repeat masking protocol: the repeat masking constraints were relaxed to allow relatively unique baits to remain in the pool of target capture baits.
To evaluate more broadly the clonal relationship of multifocal disease, custom capture baits targeting variants identified through exome sequencing of PrCa 6 were used to capture the DNA from seven additional cancer foci from that subject (Table S3).
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