HLA Genotyping using Next Generation Sequencing

Abstract

* Authors with equal contributionFrom an oncological perspective, the second most common malignancies in children are brain tumors. Despite the recent therapeutic breakthroughs in this field, concerning surgery, radiotherapy and chemotherapy alike, some cases still have poor outcomes in curability. This is especially the case in patients with high-risk histological types of tumors, and those suffering from residual, remitting and disseminated diseases. Due to the unique neuroanatomical emplacement of brain tumors and their aggressive infiltrative behavior, their total removal remains a demanding task. This can be perceived in the high rates of failure treatment and disease recurrence. Furthermore, the adjacent healthy brain tissue is inevitably damaged in the surgical process of effectively removing these tumors. Thus, stem cell transplantation may be a viable solution for the clinical management of these malignancies, as proven by various recent breakthroughs. In the current concise review, we present the role of next generation sequencing in HLA typing for stem cell transplantation in primary CNS pediatric malignancies.

Transforming growth factor β‐mediated micromechanics modulates disease progression in primary myelofibrosis

“…Quantitative RP‐PCR was performed to confirm the expression of the collagen type I and type III expression, using TaqMan PCR technology. [36‐38] For the PCR, following first‐strand synthesis, 10 μL of RT reaction mixture was used for subsequent PCR amplification by adding 40 μL of PCR master mix to the same wells. The PCR reaction mixture included 1X PCR buffer, 200 μmol/L of each dNTP and 1.25 U Taq polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA) in a final volume of 50 μL.…”

“…Quantitative RP-PCR was performed to confirm the expression of the collagen type I and type III expression, using TaqMan PCR technology. [36][37][38]…”

Human Saliva and Dried Saliva Spots as Source of DNA for PCR based HLA Typing using a Combination of Taq DNA Polymerase and AccuPrimeTaq Polymerase

“…Although NGS is cheaper than Sanger sequencing, it still remains an expensive technique for some laboratories having limited infrastructure. Also, the data analysis is time consuming, and needs big storage space and bioinformatics expertise, mainly due to the high amounts of data resulting from the sequencing experiments [33].…”

Cost-Effective TA Cloning Applied to Sanger Sequencing and HLA Allele Typing

“…It is predicted that only one out of 1081 pairs for HLA-A, 3 out of 4851 for HLA-B, and one out of 528 for HLA-C will fail to have the alleles identified in heterozygous individuals if exons 2 and 3 are analyzed together [27]. The amplified products of Class I, described here, includes the entire sequences of exon 2 and exon 3, encoding the hyper variable domains that form the peptide binding pocket of HLA class I molecules.…”