Nanopore sequencing: the missing puzzle piece in molecular identification

In this webinar, Ahmed presented his end-to-end in-field sequencing solution. Below you'll find answers to the questions he didn't have time to answer during the webinar.

1. How much does the suitcase lab cost? Was it prepared by you or is it sold as is?

The suitcase lab is around 8000 Euros and contains all necessary lab equipment. For directions on how to assemble it yourself, please refer to the following publications:

i. Abd El Wahed A, Weidmann M, Hufert FT. Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus. J Clin Virol. 69:16-21 (2015).

ii. Faye O et al. Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015. Euro Surveill. 20:44 (2015).

2. What is the power output of the generator needed to run the mobile lab? How big is the generator?

It is a 400 W solar generator, and the dimensions are 26 cm x 20.3 cm x 20.3 cm. Please see here for more information.

3. Most airlines impose a 100 Wh limit on transporting battery packs – what is the best way to transport your lab and the 400 Wh pack?

It is a gel based solar generator rather than a lithium battery, so there are no airline restrictions. I have travelled with it all over the world and experienced no problems at all.

4. What should be the minimum configuration of the high-end laptop used (in the suitcase lab)?

We used the MinIT as it is very compatible with the MinION, otherwise please check the IT requirements of MinION sequencing on the website.

5. How quantitative is your pathogen detection (suitcase lab) sequencing method?

We did not establish a quantitative sequencing approach, as the number of hits varied each run. You can check this using the ZymoBIOMICS Microbial Community Standard II (Log Distribution).

6. Can you multiplex with the Flongle? And if yes, how many samples?

Yes, up to 12 samples using Rapid Barcoding, and 24 samples with 16S sequencing.

7. Does GENEIOUS give more specific results than EPI2ME?

This depends on your databases that you have created in GENEIOUS.

8. Why do you prefer MinION Flow Cells for whole genome sequencing rather than Flongle?

First, the quality (Q) score of the data collected from the MinION Flow Cell was higher than with Flongle. Second, the number of reads was much higher using the MinION Flow Cell, which provided better genome coverage.

9. Could you please confirm which BLAST you used? MegaBLAST, Discontiguous Megablast, BLASTN?

We used Megablast.

10. Is there a cheaper alternative (perhaps freeware) for the GENEIOUS software for small research groups or NGOs that don't have money for the GENEIOUS licence?

There is a command line tool (BLAST) available from NCBI.

11. For analysis, is it possible to have an `N` symbol instead of A, C, G, or T at any position in a read, as is the case for short-read sequencing?

Yes you can have that included.

12. As a result of the sequencing experiment, is it guaranteed that all reads are in forward (or reverse) form?

There is no guarantee, you will need to check the direction.

13. How much RNA input is needed?

This is a very difficult question, a few hundred nanograms is ideal, but sometimes it is not possible to get such an amount. As few as 50 ng will work.