Gene Sequencing: Introduction, and Frequently Asked Questions (FAQs)

Introduction of Gene Sequencing

Deoxyribonucleic acid (DNA) sequencing is the process of finding out the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology (Sanger/Next-generation sequencing) that is used to find out the order of the four bases i.e. adenine, guanine, cytosine, and thymine.

Cryptococcus Sequencing

Oxford Nanopore’s MinION Sequencing Device

Frequently Asked Questions (FAQs) in Gene Sequencing

• If you have PCR products and you are requesting gene sequencing following information should be submitted-
• The size of primer used ( e.g. <500 bp).
• If you have performed electrophoresis, mention the status of bands (1 band, 2 bands, 3 bands, or more).
• Also write about the type of sequencing in which you are interested and the test charge depends on this too (forward sequencing, reverse sequencing, or both).
• You can also request sample purification.
• You can also order either Sanger sequencing or next-generation sequencing.
• Multiple bands of PCR products are bad indicators for gene sequencing.

Keynotes

• Sanger sequencing is an old method even though it is considered the “gold standard” sequencing method for validating the sequence of specific genes.

• DNA is the building block of genes.
• Oxford Nanopore’s MinION Sequencing Device is a real-time device for DNA and RNA sequencing.
• MinIon is also useful for Microbial Whole Genome Sequencing Applications.
• In general, larger DNA concentrations result in stronger signal intensities; nevertheless, sequencing issues can arise if the optimal (as shown in the table) concentrations are exceeded.
• The reason for poor or no sequence is due to-

1. lacking template or primer
2. Addition of too much primer
3. Poor quality template

• The total volume of a sample in gene sequencing should be equal to 8µl.
• The sequencing also depends on the length and purity of the PCR product/template.
• The recommended quantities of Template DNA for sequencing are as follows-

Template	Quantity
PCR product (100-200 bp)	1-3 ng
200-500 bp	3-10 ng
500-1000 bp	5-20 ng
1000-2000 bp	10-40 ng
>2000 bp	40-100 ng
Single-stranded	50-100 ng
Double-stranded (Plasmid DNA)	250-500 ng
Cosmid, BAC

Further Reading

• https://www.nature.com/articles/s41598-018-29334-5
• https://nanoporetech.com/products/minion
• https://osa.stonybrookmedicine.edu/research-core-facilities/dna-sequencing/faqs
• https://www.biotechniques.com/pcr-sequencing/what-is-dna-sequencing-answering-some-of-the-most-frequently-asked-questions/
• https://web.genewiz.com/wgs-faq