Why do we need index sequencing primer

[wisnu.wicaksono]

Hi all,

just wondering why do we use index sequencing primer and why the sequencing primer (forward or reverse) can not be used to read the whole sequence including the barcode/index. Based on illumina video ( https://www.youtube.com/watch?v=womKfikWlxM), the sequencing primer only read just before the barcode). Thank you and have a nice day.

[kmcarr]

When libraries are constructed the length of the DNA fragment between the two adapter ends is randomly distributed, determined either by random shearing (i.e. Covaris) or random tagmentation (Nextera preps). There is no way of knowing with certainty how far index 1 is away from the read 1 sequencing primer. More importantly the index is almost certainly much farther away from the read 1 primer than could reasonably be sequenced in one read; the TruSeq Nano prep kit targets insert sizes of 350 or 550bp.

Using dedicated primers for the index reads, placed immediately upstream of the index sequence is the most efficient and accurate method for sequencing the indexes.

[SylvainL]

The barcode is also in 3', meaning not the first bases which are read in order to improve the colony detection...

[wisnu.wicaksono]

Hi guys,

Thank you so much for the answer. I just want to confirm whether we will have the same conclusion or no. How about if we do amplicon sequencing that we know the lenght of amplicon for example 250 bp. Is there any possibilities that the sequencing primer can read the whole sequence include the barcode?thank you and have a nice day

[kmcarr]

Hi guys,

Thank you so much for the answer. I just want to confirm whether we will have the same conclusion or no. How about if we do amplicon sequencing that we know the lenght of amplicon for example 250 bp. Is there any possibilities that the sequencing primer can read the whole sequence include the barcode?thank you and have a nice day

But can you really know the ALL of your amplicons are 250bp? For example the 16S V4 region (515-806) is nominally 253bp (after trimming primers) but there is species dependent length variation.

If your library is designed to use standard Illumina sequencing and index primers then just set up your run to use them. They are already included in the sequencing reagents. If your are designing a library product which will require custom sequencing primers then just order the custom index primer; primers are dirt cheap compared to all the other costs in your experiment. If you will already be ordering custom read 1 and read 2 primers then you really only need to add index primer 1 which most often is just the reverse complement of the read 2 primer. (This is based on standard library designs, I can't say for certain that this will be true in your case.) For paired-end, dual indexed sequencing, index 2 is primed using the flowcell bound oligo so there is no need to order index 2 primer. (Again, based on standard designs, YMMV.)

[wisnu.wicaksono]

Hi there,

Thank you so much for your explanation, it answers all of my questions. Have a nice day