Sequencing bacteria helps us detect mutations that might be important for antibiotic resistance, virulence changes and other variations within a species. A major complication, though, is the fact that not every bacterium can be successfully grown in culture. So how can you sequence something when you can’t culture it?
A recent study points to the answer. Zheng et al. published the sequence of an unculturable alphaproteobacterium, Candidatus Liberibacter asiaticus, associated with citrus yellow shoot disease. The team extracted DNA for sequencing from an infected periwinkle tissue using the CTAB method, enriched, and later performed whole-genome amplification (WGA) using the REPLI-g Mini Kit followed by next-gen sequencing. The study generated 3.28 x 107 reads with a mean of 249 bp per read. Read the paper
So when you only have a small number of cells, whole-genome amplification can be the key to getting enough DNA for successful sequencing. The two key WGA techniques are PCR-based or multiple displacement amplification (MDA)-based. When deciding which one to use, it’s important to think about how you can get uniform amplification without bias – only unbiased amplification can give you a true picture of the mutational landscape in a cell. Numerous studies have provided evidence that MDA gives the most uniform amplification and lowest error rate because of its use of random hexamers and Phi 29 polymerase.
What challenges do you face in your lab with regard to extracting DNA from low cell numbers? How have you overcome them?