Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

AbstractAlthough various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.

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Whole-Genome Sequencing Reveals Exonic Variation of ASIC5 Gene Results in Recurrent Pregnancy Loss

Frontiers in Medicine ◽

10.3389/fmed.2021.699672 ◽

2021 ◽

Vol 8 ◽

Author(s):

Nourah H. Al Qahtani ◽

Sayed AbdulAzeez ◽

Noor B. Almandil ◽

Norah Fahad Alhur ◽

Hind Saleh Alsuwat ◽

...

Keyword(s):

Ion Channel ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Interaction Analysis ◽

Novel Mutation ◽

Direct Sequencing ◽

Recessive Mutation ◽

Whole Genome

Family trio next-generation sequencing-based variant analysis was done to identify the genomic reason on unexplained recurrent pregnancy loss (RPL). A family (dead fetus and parents) from Saudi Arabia with an earlier history of three unexplained RPLs at the ninth week of pregnancy was included in the study. Whole-genome sequencing (WGS) of a dead fetus and the parents was done to identify the pathogenic variation and confirmed through Sanger sequencing. WGS of dead fetus identifies a novel homozygous exonic variation (NM_017419.3:c.680G>T) in ASIC5 (acid-sensing ion channel subunit family member 5) gene; the parents are heterozygous. Newly designed ARMS PCR followed by direct sequencing confirms the presence of heterozygous in one subject and absence of homozygous novel mutation among randomly selected healthy Saudis. The second family with heterozygous was confirmed with three unexplained RPLs. Pathogenicity analysis of R227I amino acid substitution in ASIC5 protein through molecular docking and interaction analysis revealed that the mutations are highly pathogenic, decrease the stability of the protein, and prevent binding of amiloride, which is an activator to open the acid-sensing ion channel of ASIC5. The identified rare and novel autosomal recessive mutation, c.680G>T:p.R227I (ASIC5Saudi), in two families confirm the ASIC5 gene association with RPL and can be fatal to the fetus.

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Patients with PMD who are thoroughly screened by Genomic medicine have a considerable chance of benefiting greatly from whole-genome sequencing

10.31219/osf.io/dajft ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Direct Sequencing ◽

Genomic Medicine ◽

Diagnostic Process ◽

Whole Genome ◽

Rna Seq ◽

Tissue Samples ◽

Long Read ◽

Major Transition

It appears that the role of genetics in neurology is undergoing a major transition in the present. The scope of genomic medicine has advanced from the only realm of academic investigation to the well-established and widely accepted instrument for genetic labs. Previously, this test was reserved for the most challenging patients, but today it is being utilized as a first step in looking at rare inherited neurological disorders. Researchers and clinicians working in the field of mitochondrial medicine will need to employ new laboratory techniques and DNA sequencing technology in order to move forward with future diagnosis methods and cut down on research time. Patients with PMD who are thoroughly screened have a considerable chance of benefiting greatly from whole-genome sequencing (WGS) at the beginning of their diagnostic process. Using long-read sequencing, there is the potential to help in the discovery of new genetic causes of PMD, the resolution of phasing issues, and the advancement of RNA and mtDNA investigations by way of direct sequencing. With the use of a great number of tissue samples from patients with PMD, there are significant advantages which can greatly promote the quick implementation of this technique into diagnostic laboratories. As RNA-seq technology is introduced into diagnostic laboratories, it will serve as an accurate means to examine the entire spectrum of disease while providing support for difficult cases. The plentiful supply of tissue samples from patients with PMD further enhances the ability of RNA-seq to rapidly be adopted in these laboratories. Finally, more validation of innovative tRNA approaches will be required in order to determine the pathogenicity of this common group of mtDNA-related PMDs.

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