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Old 01-08-2018, 06:01 PM   #1
liaoyunshi
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Default How to add inline barcode to DNA fragment without PCR?

Dear all,

I want to use inline barcodes (barcode adjacent to the DNA fragment, included as the starting of the sequencing reads) for my illumina sequencing. Then I found most of current posts used this technique for short amplicon sequencing (e.g. 16S sequencing), in which the inline barcodes can be included during the PCR amplification. However, in my situation, I can only add the inline barcode after PCR and fragmentation, so I wonder if there is any kit or suggestion to add the inline barcode to the end-prep DNA fragment (I think shoule be done by ligation), or where I can design and synthesize the inline barcodes (essentially, short double strand DNA)?

Thanks.
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Old 01-08-2018, 07:19 PM   #2
luc
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Hi liaoyunshi,
Adding inline barcodes by ligation requires more effort (synthesis of two partially complementary oligos for each barcode) than adding truseq-style barcodes. Is there a special reason why these should be inline?
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Old 01-08-2018, 09:20 PM   #3
liaoyunshi
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Hi luc,

I think the truseq-style barcodes you mentioned is that kind of "multiplex index" within the adapter? The reason why we do not want to use such common way is that we want to conduct such multiple DNA as one single illumina sample, which would be then joined into a even huge "multiplex barcode" run (hope you can understand my poor description). Thus to differentiate those multiple DNA after sequencing, we need to include some identifiers within the resulting read. Given that the long length of the whole set truseq-style barcodes, it will occupy much of the fixed resulting read length. So we just wonder if it is feasible to self-incorporate inline barcode besides the inserted fragments to save the read length.

And do you know if there is any kits can provide us those inline barcodes that are able to be ligated? Or I even need to generate inline barcodes ab initio as double strand oligos and then try very hard to ligate them into the raw DNA fragments?

Thanks.
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Old 01-09-2018, 04:17 PM   #4
luc
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Hi liaoyunshi,

sorry, I do not understand what you are trying to achieve with the inline barcodes.
Working with the tru-seq style barcodes is preferable for all applications I can think off.

I have attached an old protocol with inline barcodes written by the Comai lab at UC Davis.

https://drive.google.com/file/d/10QD...ew?usp=sharing
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barcode sequencing, illumina, next-gen seq

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