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Next, TFBS-ID close (≤5 KB) to the transcription start site (TSS) of known human genes (as defined by the Reference Sequence set) were selected.
For each element family, the potential number of full-length copies is estimated by the division of the estimated size of total members of the element in the genome by the reference sequence length.
The 4qA-L fragment was sequenced and all 30 CpGs predicted by the reference sequence were accounted for in 100% of the analyzed sequences from these clones, confirming the specificity of the reaction for the distal 4qA-L-derived D4Z4.
To test our methodology, we identified a collection of 99 negative control junctions; these junctions are predicted to be constitutively spliced given that they are present in every mRNA isoform curated by the Reference Sequence Database (RefSeq) [ 21].
However, because the transcriptomes are incomplete, even for well-studied species such as human and mouse, analysis of RNAseq data is restricted by the reference sequence, and requires another calculation to identify novel transcripts.
Since the O. kenojei mitochondrial genome has been annotated by the Reference Sequence Project at the NCBI, these conflicts could be readily resolved by examining a tRNA gene in this genome.
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Protein sequences for B. bovis were obtained by searching the reference sequence collection of GenBank for species name 'Babesia bovis'.
The upstream sequence breakpoints for each deletion were determined by aligning the reference sequence with the sequences generated using the forward primers in the sequencing reaction.
Downstream breakpoints were determined by aligning the reference sequence with the reverse complements of the sequences generated using the reverse primers.
Finally, SNPs in the ELOVL6 gene were manually identified by comparing the reference sequence with mapped reads with the Integrative Genomic Viewer (IGV v.2.1) [ 29, 30].
However, note that our focus is on re-sequencing studies, and if we have even a crude idea on the genomic coordinates of the BACs, we can approximate that missing information by using the reference sequence.
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by the promoter sequence
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by the chemisorption sequence
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by the opening sequence
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by the scramble sequence
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