"Illumina’s “indexing” system differs from other sample barcoding methods for high-throughput sequencing in that the barcodes (“indexes”) are placed within one of the adapters rather than being directly attached to the ends of template molecules"
recently I have read about illumina sequencing technic. The question comes that is it necessary to put index in the middle of the primers when doing PCR.
If we send our PCR sample to sequencing companies with index directly attached to the ends, will it do harm to the results?
The within-adaptor index is sequenced separately from the DNA insert (and has an immediately preceding sequencing primer of its own). If we were to stick these indices into the DNA insert than we would be consistently taking away high-quality bases from the primary sequencing effort. We can't place the index at the end of the insert, because per-base quality rapidly decreases at the ends of the read as individual fragments in the cluster become more and more asynchronous (which is why we tend to trim the ends). By giving the index its own sequencing primer we can guarantee that those base calls will be of higher quality while not shortening the amount of DNA insert we can effectively sequence, yet we can still assign those index reads to the sequence reads because they are from the same physical location on the flowcell.
You can of course also place barcodes at the end of your DNA insert, but be sure to place it on the P5 end so that sequence quality is maximized, although these won't be automatically demultiplexed. In our case, we multiplex more individuals than there are available Illumina indices, so we use combinatorial indexing with a barcode at the P5 end of the insert as well as the Illumina index in the adaptor at the P7 end.
The within-adaptor index is sequenced separately from the DNA insert (and has an immediately preceding sequencing primer of its own). If we were to stick these indices into the DNA insert than we would be consistently taking away high-quality bases from the primary sequencing effort. We can't place the index at the end of the insert, because per-base quality rapidly decreases at the ends of the read as individual fragments in the cluster become more and more asynchronous (which is why we tend to trim the ends). By giving the index its own sequencing primer we can guarantee that those base calls will be of higher quality while not shortening the amount of DNA insert we can effectively sequence, yet we can still assign those index reads to the sequence reads because they are from the same physical location on the flowcell.
You can of course also place barcodes at the end of your DNA insert, but be sure to place it on the P5 end so that sequence quality is maximized, although these won't be automatically demultiplexed. In our case, we multiplex more individuals than there are available Illumina indices, so we use combinatorial indexing with a barcode at the P5 end of the insert as well as the Illumina index in the adaptor at the P7 end.
Thanks a lot for your detailed reply. I have some other questions.
1. Do we still need to add protective bases other than 6 bases index if we send our sample to sequencing company which said they add additional sequencing adaptor to the indexed DNA insert?
2. I've heard about P5 and P7 primers, is it possible we use self-designed index primer (with index on the one end, without protective bases), rather than the exact P5 and P7 primer provided by illumina (in case that the company add additional sequencing adaptor)?
Looking forward to your reply
Hi Hao,
The principle of Illumina Sequencing may help you reach the answer of your questions.
1. Generally, it is not necessary to add protective bases. You just send you samples to sequencing company, and they will add the given adaptor and index(adaptor and index were provided by Illumina company).
2. P5 and P7 were also provided by Illumina company. This two primers were used to attach to Flow Cell and amplification primer design.
Best Regards!
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