Dual-End Barcoding for 16S rRNA Primers: Using Golay Barcodes on Both Forward and Reverse Primers

General Discussion
1

Hello,

Our lab is working on developing a 16S rRNA sequencing protocol, and we would like to incorporate barcodes on both the forward and reverse primers. Currently, we are following the EMP protocol, where only the forward primer (515F) contains a Golay barcode.

I was wondering:

  1. Can we simply take the next 20 Golay barcode sequences from the forward primers and insert them in the appropriate position within the reverse primers to achieve dual-end barcoding?
  2. Will this approach affect demultiplexing in QIIME 2, or are there any potential issues with assigning reads correctly if both primers contain barcodes?
  3. Are there any recommended guidelines or considerations for designing barcoded reverse primers for 16S sequencing?

Any advice or experience with dual-end barcoding for 16S rRNA sequencing would be greatly appreciated!

Thank you!

3

Hi,

we recently developed such a dual-index library preparation protocol based on the EMP primers and unique barcodes for 16S (515/806 primers) as well as for ITS (BITS/B58S3 primers)! :sparkles:
It is currently under review, but can already be accessed here:

HighALPS: Ultra-High-Throughput Marker-Gene Amplicon Library Preparation and Sequencing on the Illumina NextSeq and NovaSeq Platforms
Lena Flörl, Paula Momo Cabrera, Maria Domenica Moccia, Serafina Plüss, Nicholas A.Bokulich
bioRxiv 2024.10.10.617643; doi: https://doi.org/10.1101/2024.10.10.617643

To answer your questions

  1. Yes - we also used the Golay barcodes for the forward and reverse primers and then in silico checked whether they form dimers. We provide such forward and reverse constructs with adapter, barcode and primers in the supplementary material (which is not linked in bioRxiv yet, but I can share it with you via email). :hammer_and_pick:

  2. Most often the demultiplexing is already done on the sequencing platform. When you load the library, you provide the custom barcode list and get the demultiplexed reads in return (note, that Illumina barcodes are shorter, so this needs to be adjusted in the sequencing run settings). But also with QIIME2 this is not an issue, and you can simply demultiplex with your sample & barcode list! :partying_face:

  3. Like I mentioned, it's important to check whether the forward and reverse primer construct does not dimerise. But you can also read more on this in the publication linked above. :arrow_up:

I hope this is of interest or helpful to you! Please feel free to ask if you have any additional questions. :blush:

Best,
Lena

3 Likes
4

Hi Lena,

Wow, thank you so much for helping me. If possible, I would love to see the forward and reverse constructs in supplementary material. Please, send them to the immrosadi00@gmail.com.

Thank you in advance

Best,
Imam

5

Hi, I just sent it to you - good luck with it! :mailbox:

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6

Thank you so much!! I got it. It really help me.

1 Like