Libraries with unamplified DNA show more uniform coverage across Chromosome I compared with amplified libraries.
Within the T7 Trinuc population, the propensity to form β-sheets was the greatest, followed by β-turns, and then α-helices, and the percentage of the three secondary structures was more uniform than in the NNK-randomized libraries.
Kozarewa et al. [ 24] demonstrated that using a PCR amplification-free enrichment step that relies solely on cluster amplification on the sequencing platform for the enrichment of the library, resulted in a more uniform distribution of reads.
Use of such genetic codes is beneficial in that the reduction in amino acid codon number grants each amino acid a more uniform chance of incorporation into the peptide library, and that the reduction in stop codon number minimizes the occurrence of truncated peptides within the library.
Therefore, to obtain more uniform sequencing coverage, we prepared all subsequent libraries from polyA-enriched RNA.
A distinct pattern was found for genes never found in the EST library ("weakly expressed"), for which codon usage is more uniform, with no codons strongly dominating.
As each amino acid is granted an equal opportunity for incorporation into the phage library DNA, the trinucleotide cassette randomization method allows for more uniform amino acid frequencies as compared to degenerate codon randomization methods.
Therefore performing manual DNA extraction or preparing multiple libraries with independent automated DNA extractions may result in more uniform sequencing and fewer missing values when the GBS method of Elshire et al. (2011) [ 37] is applied to linkage map construction.
However, the more uniform the initial distribution is, the better the final performance of the trained classifier will be, so the design of a uniform library is one of the important considerations for the future experimental implementation of the classifier.
Ross et al. previously reported that for human samples, PCR-free library construction could increase the relative coverage for high AT regions from 0.52 to 0.82, resulting in a more uniform coverage [ 22].
