Next generation sequencing (NGS) is a wonderful tool for identifying waterborne pathogens. But I was wondering what are the challenges we face in NGS in terms of identifying the correct pathogen from a mixture of samples and in terms of identifying from a environmental sample with very low copy number?
Hi,
Of course, all NGS approaches (library preparation methods) come with a certain degree of drop-outs. Usually, as lower your input material, as higher the chance that the low abundant RNA/DNA molecule would not be caught and proceeded till the sequencable piece of library. Such, in usual bulk RNA-seq, when you start with a lot of input material, you end up detecting up to 40k transcripts (referring to human or mouse), while in single-cell RNA-seq you can barely reach 3-6k transcripts detected.
Intuitively, deeper sequencing results in the higher amount of transcripts detected, though at some point you reach a plateau in the unique_transcript/total_reads graph (=saturation curve), which represents the redundant PCR duplication of the transcripts, but not the appearing of new ones.
Overall, you need to pick the right library preparation kit, use high amount of input material and sequence deep. This will result in the high sensitivity.
Best,
Michail
In addition to Michail Yekelchyk just wanted to add the limitations of the databases in the identification too.
- Badges
- Science method