Hi Folks,
I'd like to sequence a few 16S rRNA amplicons in one lane of an Illumina flow cell. I need to barcode my samples and would like a bit of advice. I have already read the threads in this forum discussing barcoding, but I guess I'm in need of more specific advice and would like to know if there is anyone out there with recent experience. A recent PNAS paper used a barcoding protocol which seems suitable (see Fig 1 of attachment). As I understand it, these researchers prepared their libraries as follows:
1) Made composite PCR primers consisting of (5`--> 3´) the Illumina adapter+the barcode+linker+16S reverse primer. Same for the forward primer but without the barcode.
2) Amplification products thus had Illumina adapters and barcodes
3) Sequenced in the GAIIx using custom sequencing primers (Read1, Read2 and Index Sequencing Primer). The use of custom sequencing primers, as I understand it, allowed them to get 100 bp reads that covered the insert (product of interest) and thus read length was not sacrificed to sequencing the 16S primer.
I have thought of hopefully replicating this, perhaps designing the barcodes with barcrawl and the linkers with PrimerProspector. However, I believe there might be some pitfalls of which I'm not aware. Besides, I am not 100% confident if I understand how this library was prepared.
I'd enormously appreciate help/comments from the community! Thanks!
All the best,
A
I'd like to sequence a few 16S rRNA amplicons in one lane of an Illumina flow cell. I need to barcode my samples and would like a bit of advice. I have already read the threads in this forum discussing barcoding, but I guess I'm in need of more specific advice and would like to know if there is anyone out there with recent experience. A recent PNAS paper used a barcoding protocol which seems suitable (see Fig 1 of attachment). As I understand it, these researchers prepared their libraries as follows:
1) Made composite PCR primers consisting of (5`--> 3´) the Illumina adapter+the barcode+linker+16S reverse primer. Same for the forward primer but without the barcode.
2) Amplification products thus had Illumina adapters and barcodes
3) Sequenced in the GAIIx using custom sequencing primers (Read1, Read2 and Index Sequencing Primer). The use of custom sequencing primers, as I understand it, allowed them to get 100 bp reads that covered the insert (product of interest) and thus read length was not sacrificed to sequencing the 16S primer.
I have thought of hopefully replicating this, perhaps designing the barcodes with barcrawl and the linkers with PrimerProspector. However, I believe there might be some pitfalls of which I'm not aware. Besides, I am not 100% confident if I understand how this library was prepared.
I'd enormously appreciate help/comments from the community! Thanks!
All the best,
A
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