Targeted Next-Generation Sequencing
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Questions and Answers

What is the primary source of the human genome 19 reference used in this study?

Where can the list of inherited diseases associated with the Ion AmpliSeq™ Inherited Disease Panel target gene, including cystinosis disease, be found?

What type of consent was obtained from the patient and the future couple for molecular genetic testing?

Written informed consent

What is the purpose of agarose gel analysis of the RT-PCR amplification products in this study?

<p>To analyze the RT-PCR amplification products of the patient and the couple</p> Signup and view all the answers

Where can the complete datasets used and/or analyzed during this study be obtained from?

<p>The corresponding author, upon request</p> Signup and view all the answers

What is the primary goal of targeted parental carrier testing in couples with a family history of cystinosis?

<p>To identify carriers of the disease-causing mutation and provide reproductive options to prevent or minimize the risk of having children with cystinosis.</p> Signup and view all the answers

What is the most common and serious clinical manifestation of cystinosis, and at what age does it typically occur?

<p>Renal failure, typically occurring before the age of 20.</p> Signup and view all the answers

What is the genetic basis of cystinosis, and how is it inherited?

<p>Cystinosis is an autosomal recessive disorder caused by mutations in the CTNS gene, leading to a lysosomal storage disease.</p> Signup and view all the answers

What reproductive options are available to couples with a family history of cystinosis, and how can they be implemented?

<p>Couples can consider preimplantation genetic diagnosis, prenatal testing, or targeted premarital genetic testing to reduce the risk of having children with cystinosis.</p> Signup and view all the answers

What is the significance of identifying the pathogenic mutation in the CTNS gene in the patient, and how can it inform genetic counseling?

<p>Identifying the pathogenic mutation enables accurate genetic counseling, carrier testing, and reproductive planning for the family.</p> Signup and view all the answers

Study Notes

Library Preparation and Sequencing

  • The library was quantified using the Qubit dsDNA HS analysis kit on a Qubit 2.0 fluorometer.
  • Emulsion PCR was performed on the OneTouch2 system using the Ion PGM HI-Q OT2 kit.
  • Template enrichment was done using Ion OneTouch ES, and Ion 318 Chip v2 was prepared for loading.

Targeted Next-Generation Sequencing

  • Libraries were prepared using Ion AmpliSeq Library Kit v2.0 for the patient and two other patients affected by hereditary diseases.
  • The panel contains three primer pools, and amplification was performed using the Ion Ampliseq HiFi Mix.

RNA Extraction and RT-PCR

  • Total RNA was isolated from leukocytes using the RNeasy Mini Kit.
  • cDNA was performed using Superscript III Reverse transcriptase.
  • cDNA amplification of the CTNS gene was performed to confirm the deletion using specific primers.

Sanger Sequencing

  • The reaction products were analyzed by 1% agarose gel electrophoresis.
  • Sanger sequencing was done using dye terminator chemistry and run on an automated sequencer.

Data Availability and Ethics Approval

  • The datasets used and/or analyzed during the study are available from the corresponding author upon request.
  • Written informed consent for molecular genetic testing was obtained from the patient and the future couple.
  • The study was approved by The Ethics Committee of the National Institute of Health in Rabat, Morocco.

Cystinosis

  • Cystinosis is a rare autosomal recessive lysosomal storage disorder described in 1903 as a familial cystine accumulation.
  • It's a systemic disease, and the kidney involvement is the most serious clinical event.
  • In more than 90% of patients, it drives them to end-stage renal disease before the age of 20.

Genetic Counseling

  • Genetic counseling and targeted parental carrier testing, prenatal, and preimplantation genetic diagnosis in subsequent pregnancies, and targeted premarital genetic testing in at-risk couples are recommended.
  • This is because the couple has a family history of a severe form of cystinosis.

CTNS Gene

  • The CTNS gene is associated with cystinosis.
  • The Ion AmpliSeq Inherited Disease Panel can provide a high degree of multiplexed target selection of all exons and some gene introns associated with more than 700 unique genetic diseases.
  • The sequence data analysis detected a potential deletion spanning exons 4 and 5 in the CTNS gene.

Case Study

  • The patient, a 22-year-old girl, was diagnosed with cystinosis in early childhood.
  • She has a severe form of cystinosis, which progressed towards renal failure at the age of 6 years old.
  • Her sister's husband and the couple (V-1 and V-2; Fig. 1) came to the center for genetic counseling.

Molecular Studies

  • The authors used Invitrogen kit to extract genomic DNA from peripheral blood lymphocytes.
  • The Qubit dsDNA HS Assay Kit was used to quantify DNA with Qubit Fluorometer.
  • The sequence data analysis detected a potential deletion spanning exons 4 and 5 in the CTNS gene.
  • The candidate variants were obtained when variant frequency at a given position is ≥20% and variant coverage is ≥20×.

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Description

Quiz about preparing libraries for targeted next-generation sequencing, including quantification, emulsion PCR, and template enrichment. Learn about the necessary steps and tools for sequencing.

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