Design, construction, and validation of a modular library of sequence diversity standards for polymerase chain reaction

Abstract

Methods to measure the sequence diversity of PCR-amplified DNA lack standards for use as assay calibrators and controls. Here, we present a general and economical method for developing customizable DNA standards of known sequence diversity. Standards ranging from 1 to 25,000 sequences were generated by directional ligation of oligonucleotide "words" of standard length and GC content, and then amplified by PCR. The sequence accuracy and diversity of the library were validated using AmpliCot analysis (DNA hybridization kinetics) and Illumina sequencing. The library has the following features: (1) pools containing tens of thousands of sequences can be generated from the ligation of relatively few commercially-synthesized short oligonucleotides; (2) each sequence differs from all others in the library at a minimum of three nucleotide positions, permitting discrimination between different sequences by either sequencing or hybridization; (3) all sequences have identical length, GC content, and melting temperature; (4) the identity of each standard can be verified by restriction digestion; and (5) once made, the ends of the library may be cleaved and replaced with sequences to match any PCR primer pair. These standards should greatly improve the accuracy and reproducibility of sequence diversity measurements.

Microarray Generation of Thousand-Member Oligonucleotide Libraries

“…Similarly, significant skewing has previously been reported in Solexa sequencing of a PCR-amplified synthetic oligonucleotide library [38].…”

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists

“…For sorted T-cell subpopulations, which have more uniformity in their clone frequency, the absolute number of sequences of a given VJ family in the sample was calculated using quantitative standards as described. 18 This value was then used to estimate the total number of sequences in the sample by multiplication with measured parameters of VJ family usage as described previously. 17,19 The error resulting from selection of individual VJ families was assessed by measuring the reproducibility of measurements of the same samples using different VJ families (supplemental Figure 3, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).…”

“…5,14 Although these data have been used to suggest that TCR specificities are lost from the repertoire during the course of HIV disease, they could simply reflect the preferential expansion of selected T-cell clones in HIV-infected persons. AmpliCot, an assay taking advantage of DNA hybridization kinetics and amenable to the evaluation of very large numbers of diverse sequences, [17][18][19] enabled the quantitative assessment of TCR repertoire diversity of human blood or cell samples. AmpliCot has been validated, using both oligonucleotide libraries and titrated naive T cells, to make reproducible distinctions between samples with 2-fold differences in diversity.…”

“…AmpliCot has been validated, using both oligonucleotide libraries and titrated naive T cells, to make reproducible distinctions between samples with 2-fold differences in diversity. [17][18][19] Interassay reproducibility for replicate samples collected in this study is shown in the supplemental Appendix. For samples containing single types of cells with relatively similar frequencies of TCR genes, it is possible to use standards to estimate the absolute number of unique TCR rearrangements in the sample.…”

“…Here we apply the quantitative AmpliCot method [17][18][19] to a cross-sectional study of HIV-infected and -uninfected subjects to clarify the impact of HIV disease on the TCR repertoire. One challenge with quantitative measurements of TCR diversity is that they are dependent on the nature of the sample used.…”