Q about asymetrical barcodes

[splaisan]

Dear Vladimir,

We work with assymetrical barcodes in order to multiplex a large number of samples on our Sequel-IIe (inspired from the 16S pipeline and using the same layouts).

I read in your usage page that assymetrical barcodes are not currently supported. Very sad! This marks the end of my exploration as I cannot change that in the wet lab and multiple samples always share the same barcode on either end.

Do you have an estimate on when this could be implemented?

Thanks in advance

[vmikk]

Hello Stéphane!

Thank you for reaching out and sharing your feedback. I understand the importance of this feature, especially when you're multiplexing a large number of samples.

You're right; as of now, we don't support asymmetric barcodes. However, as a workaround, you can pre-demultiplex your data outside of NextITS and then proceed with the analysis. When doing this, you would set --input as a directory containing your fastq files and also specify --demultiplexed true.

While I don't have a concrete estimate for when asymmetric barcode support will be implemented, I genuinely believe it's a feature worth adding. We just need to allocate the time and resources to make it happen. Your feedback is invaluable in helping us prioritize such features.

On a side note, I'm curious about the nature of your barcodes. Are they "purely asymmetric" (i.e., only different barcodes on each end, like A-B), or do you also use symmetric combinations (like A-A)?

[splaisan]

Hello Vladimir,

This is great news, especially since I get my reads already demuxed from the Sequel-IIe. I try this as soon as I receive the first run. I cant wait to see the first results.

The barcodes are the exact same used in the PB 16S kit and are purely assymetrix indeed, you can find them here (https://www.pacb.com/wp-content/uploads/Asy_M13_BUP_Sequence_384plate.xlsx). For ITS, I used the first 2 plates combination only (1 forward set and 2 reverse sets), which can be extended with a second reverse set to reach 384 plex (we normally go only until 192 plex).

Best regards

[vmikk]

NextITS now supports asymmetric barcodes. Please use the --lima_barcodetype parameter to specify the barcode type. The supported options are single, dual_symmetric (default), dual_asymmetric, and dual (for a mixture of symmetric and asymmetric barcodes). For dual_asymmetric and dual types, barcodes should be specified as follows:

>Run01__Sample1
ACTCATCTTCCA...ACTCATCTTCCA
>Run01__Sample2
AGCAATCGGTAT...AGTAATAGGGAT
>Run01__Sample3
AGTCCACTGGTA...GACGGACTGGTG