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The raw sequence throughputs obtained were consistent with the ones expected for the portion of the sequencing area used for each instrument, as specified by each manufacturer.
Considering, however, that the same relative sequencing surface was used for all the machines (1/8 of the total sequencing area), mapping of Roche 454 raw reads produced an average coverage of ∼770x/base, of Illumina GA reads ∼4,000x/base, and of ABI SOLiD reads ∼26,000x/base.
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Despite the limitations on amplicon size imposed by 454 sequencing, areas of no or limited sequence heterogeneity that were suitable for primer design could be identified in the regions immediately proximal and distal to the V3-coding sequence.
In addition, both sequence preparation methods are poor at sequencing areas of substantially lower %GC (< 30%).
Interestingly, the latter sequence area of rMal d 1 contained an unpaired cysteine residue C107.
The nucleotide sequence area is from 982 to 1928 of FJ609231, and looks like a rearranged FJ223605.1.
A phylogeny, based on neighbour joining of the amino acid sequence area from C1 to C8+1, is given in Fig. 3.
This could be helpful to maintaining the conservative sequence area around the binding sites of amino acids that shape the spatial structure of the functional unit.
To validate the microarray data analysis, we performed semi-quantitative RT-PCR using primers designed in the exon sequence area matching the four probes.
The regulatory elements were identified within a sequence area previously shown to mediate drug response via PXR and CAR and to contain a binding site for HNF-4α (Gnerre et al. 2004).
The results presented below are not affected by these un-sequenced nucleotides because AFLPs, gene locations and their distances obviously refer only to sequenced areas, with un-sequenced nucleotides generally being clustered in large regions.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com