Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles.
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ABSTRACT: Objective
We aimed to simplify our fetal RHD genotyping protocol by changing the method to attach Illumina's sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its reliability and robustness by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors.Results
Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal RHD genotyping for East Asian populations into clinical practice.
SUBMITTER: Hori A
PROVIDER: S-EPMC8474863 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Introduction: Since anti-D immunoprophylaxis given to D-negative pregnant women is a blood product, blood donations have an impact on the availability of prophylactic doses. The Pan American Health Organization reported, in June 2017, that less than half of blood donors are volunteers in Latin America and the Caribbean. In these countries, guidelines for use of anti-D prophylaxis are still controversial. The aim of this study was to demonstrate the convenience of a simple and cost-effectivene non-invasive prenatal diagnostic assay for anti-D prophylaxis optimization in multiethnic populations. Methods: Cell-free fetal DNA from plasma samples of D-negative pregnant women were analyzed by real-time PCR for simultaneous amplification of sequences of exons 5 and 10 of the RHD gene. Fetal RHD genotype was determined in 111 pregnant women. Neonates' phenotype was determined 72 h after birth. Results: Genotyping predicted fetal phenotype with 100% accuracy. Prenatal diagnosis showed 78% RHD-positive and 22% RHD-negative neonates. Conclusion: We demonstrated that, beyond the large genetic variation of the Rh system and the numerous D variants present in multiethnic groups, non-invasive fetal RHD genotyping using two sequences of the gene can be enough for clinical application in an admixed population. This robust technique of simple implementation allows to determine fetal RHD in maternal blood with high sensitivity, specificity, and accuracy. The introduction of fetal RhD genotyping as part of an antenatal screening program constitutes a reliable manner to optimize anti-D prophylaxis; however, it has not been implemented so far in most American countries.
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2014-06-24 | E-MTAB-2566 | biostudies-arrayexpress
Project description:BackgroundNext-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol).A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure.ResultsA pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias.ConclusionsThe single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.
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