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The best alignment to a sequenced Micromonospora CBM family 33 protein (NCBI reference sequence WP_007071991.1) resulted in 70% identity.
Bismark fully bisulfite converts sequence reads and maps each to bisulfite converted versions of the reference genome, with only reads producing a unique best alignment to a region being kept.
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As earlier common computational methods for identifying precursor genes, a gene with the best alignments to a chimeric mRNA was considered as the precursor gene [ 7].
This requirement forces the best alignment to match a majority of the probe sequence, since BLAT produces many spurious short alignments with 100%percentt identity.
Partially-mapped reads are defined as high-quality full length (76 bp) reads where the best alignment to targeted regions is a perfect match less than 76 bases in length.
Using an informatics pipeline that allowed up to two mismatches in a read to map to the reference, we found that 43.9% of the B73 fragment reads had a single best alignment to the reference, 46.7% could be aligned to multiple positions, and 9.3% could not be aligned to the reference at all.
First, reads were required to have a best alignment to the reference assembly with at least 3% more identical nucleotides than the second-best alignment.
Twenty-six (54%) of the non-conserved miRNAs had a best alignment to Arabidopsis sequences, of which two predicted target genes were annotated only as expressed sequences.
Over 78% of the mapped reads included at least one mismatch and 22% of the mapped reads have four or five mismatches in their best alignment to the reference, indicative of a high machine error rate.
Despite having the best alignment to work with, we still see a fairly large difference between the variant callers, which is likely attributable to the different algorithms they employ).
Cleaned reads were mapped to the maize B73 version 2 pseudomolecules (AGPv2; http://ftp.maizesequence.org/) (Schnable et al. 2009), using Bowtie version 0.12.7 (Langmead et al. 2009), allowing up to two mismatches and requiring a single best alignment to nuclear DNA.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com