Primer barcoding

[Kutay Ozturk]

Hi, 

I am considering whether LAMP-Seq can be utilized in detection of other viruses on other hosts. but I am just getting familiar with the system and I have some questions I would appreciate if you are able to answer. I looked through the manuscript and the supplementary material for designing barcoded primer sequences. I see that in Supplementary Note 1, factors to take into account in barcoding and detailed analyses are presented, but I could not see the protocol for designing the barcoded primer sequences, which software/code was used etc. Also, would it be possible that the inclusion of different barcode sequences may impact the specificity, sensitivity, and overall efficiency of the amplification, or is it safe to assume that all 10,000 barcode sequence-containing primers would be equally efficient?

Thank you

[LAMP-Seq]

Hi, 

The code for designing the error-correcting barcodes are available at: https://github.com/feldman4/dna-barcodes. 

As mentioned in the manuscript "Next, pooling 480 barcoded FIP primers, performing RT-LAMP reactions in four replicates, and sequencing the barcode distribution in resulting products revealed that ~5% of barcode sequences perform poorly or even fail to engage in LAMP-Seq (Fig. 2F). Investigating potential sequence determinants that could guide optimized barcode design, we observed that the least efficient barcode primers displayed a marked enrichment for a GTCC motif or truncations thereof, especially towards the 3’ end of the barcode (Fig. 2F, inset). As this motif is the reverse complement of the 3’ end of the FIP primer, we hypothesize it could sequester the 3’ end by forming an intramolecular structure, thus inhibiting elongation of the primer, and should be avoided. Following this rule, we designed >6,000 barcoded FIP and BIP primers as well as provide 240 validated barcoded FIP primers for application of LAMP-Seq (Table S1)." So, yes, the barcode sequences can lead to differing overall efficiencies of amplification. 

Hope this helps.