Non-invasive prenatal testing of beta-hemoglobinopathies using next generation sequencing, in-silico sequence size selection, and haplotyping.

Aim: To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing.

Methods: We applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF).

Noninvasive Prenatal Test for β-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

Background: Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the β-hemoglobinopathies to reduce the risk to the fetus.

Correction: Shelly Y. Shih; et al.; Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples. Genes 2018, 9, 49.

The authors wish to make the following change to their paper [1][...

Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples.

The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library.

A phylogenetic approach for haplotype analysis of sequence data from complex mitochondrial mixtures.

Massively parallel (next-generation) sequencing provides a powerful method to analyze DNA from many different sources, including degraded and trace samples. A common challenge, however, is that many forensic samples are often known or suspected mixtures of DNA from multiple individuals. Haploid lineage markers, such as mitochondrial (mt) DNA, are useful for analysis of mixtures because, unlike nuclear genetic markers, each individual contributes a single sequence to the mixture.

Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions.

Aim: To apply massively parallel and clonal sequencing (next generation sequencing or NGS) to the analysis of forensic mixed samples.

Methods: A duplex polymerase chain reaction (PCR) assay targeting the mitochondrial DNA (mtDNA) hypervariable regions I/II (HVI/HVII) was developed for NGS analysis on the Roche 454 GS Junior instrument. Eight sets of multiplex identifier-tagged 454 fusion primers were used in a combinatorial approach for amplification and deep sequencing of up to 64 samples in parallel.

A twin study of mitochondrial DNA polymorphisms shows that heteroplasmy at multiple sites is associated with mtDNA variant 16093 but not with zygosity.

The mitochondrial theory of ageing proposes that damage to mitochondria and diminished mitochondrial DNA (mtDNA) repair are major contributors to cellular dysfunction and age-related diseases. We investigate the prevalence of heteroplasmy in the mtDNA control region in buccal swab and blood derived samples for 178 women from the TwinsUK cohort (41 DZ pair 39 MZ pairs, 18 singletons, mean age 57.5 range 28-82) and its relationship to age, BMI and fasting insulin and glucose serum levels.

DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras.

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA.

Mitochondrial DNA typing screens with control region and coding region SNPs.

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens.

Identification of human remains by immobilized sequence-specific oligonucleotide probe analysis of mtDNA hypervariable regions I and II.

Aim: A rapid analysis of mitochondrial DNA (mtDNA) sequences with an array of immobilized sequence-specific oligonucleotide (SSO) probes was tested on 18 skeletal elements recovered from mass graves in Croatia, which could not be genotyped with common forensic nuclear DNA systems (PM+DQA1 and short tandem repeat analysis).

Methods: We used duplex polymerase chain reaction (PCR) amplification of the mtDNA hypervariable regions I and II (HVI and HVII) (444 bp and 415 bp amplicons, respectively) and subsequent linear array typing, which targets six polymorphic regions and two additional sites within the human mtDNA HVI and HVII. The remaining amplified products were subjected to direct sequence analysis to obtain complete sequence information for the targeted HV regions.