Good index sequences for unique dual indexing?

[Innovelty]

Still a little confused

I'm a bit confused about how to accomplish the dual-indexing using the same index on each end. My workflow ligates an adapter, and then uses indexing primers in PCR to get flow-cell adapters and indices onto the molecules. When I'm single indexing, this involves a "universal forward primer" and a unique reverse primer with the index. I can't just use the reverse primer without the universal forward primer, can I? I won't get indices on both ends, will I?

This seems awfully elementary, I know, but it's hard to keep track of exactly what protocol people are talking about doing. I 100% understand using two unique index primers for your library, out of, say, Illumina's TruSeq dual-indexing kit. But I don't understand how people are suggesting using the same index on both ends. You've got to be using custom oligos, right, with a forward and reverse primer that contain the same index region?

Are some sequencing cores now stocking indexing primers like this that customers can purchase? Because I sure as heck cannot afford 96 pairs of custom Illumina primers.

[apredeus]

You can use dual indexes (or dual unique indexes) even with single end reads. Here is a link to Illumina's index sequencing overview which describes all possibilities of indexing, sequencing and instruments.

Thank you very much! This is extremely useful.

[kmcarr]

Along the same lines, and rather than multiplying threads - are there any comprehensive reviews of all the indexing strategies available for Illumina?

And what would be a most reliable indexing strategy for single-end sequencing on Hiseq3000/Hiseq4000? Some long custom indexes, maybe?

I would appreciate any input or pointers to discussions on other sites or here.

You can use dual indexes (or dual unique indexes) even with single end reads. Here is a link to Illumina's index sequencing overview which describes all possibilities of indexing, sequencing and instruments.

[nucacidhunter]

I have not seen one. Generally it includes:
1- Indexes included in adapter (i5 and i7) which is Illumina’s standard method
2- Inline barcode where one can include a barcode sequence at the start of one or both of reads. Barcode can be fixed or variable length for different samples to increase sequence diversity if required.
3- Combinatorial indexing which is using inline barcode and standard index for samples

To minimise read miss-assignment due to index hopping one can dual index libraries with unique i5 and i7 sequences. Longer index will be inefficient in prevention or identification of index hopping.

[apredeus]

Along the same lines, and rather than multiplying threads - are there any comprehensive reviews of all the indexing strategies available for Illumina?

And what would be a most reliable indexing strategy for single-end sequencing on Hiseq3000/Hiseq4000? Some long custom indexes, maybe?

I would appreciate any input or pointers to discussions on other sites or here.

[jlei_face]

As you have probably already seen, Illumina will be working with IDT to create these UDI's. I know that IDT already has 96 unique indices, 8 nt long, as I have used them in the past. Instead of waiting for Illumina to release their kit it might be worth contacting IDT directly...

[MU Core]

I've been told that Illumina will soon (~1-2 months) be releasing a kit with 96 dual-unique indexes.

[kmcarr]

I have heard unconfirmed reports that both Kapa and Bioo are planning on offering sets of 96 dual-unique indexes.

[jwfoley]

After the index-hopping scandal (see Illumina's white paper) it sounds like the field is moving in the direction of unique dual indexing, i.e. dual indexing where every index combination is unique, so that no two libraries have the same P7 index and no two libraries have thes same P5 index. The means that instead of 12 versions of the P7 adapter and 8 versions of the P5 adapter, you'd need 96 versions of P7 and 96 versions of P5. This is an expensive investment for homebrew library protocols.

Does anyone know of experimentally validated index sequences for 96-plexity?
It would be nice to keep the 2 x 8 nt index-read lengths so we don't have to steal cycles from the actual target sequence. I see Bioo has a set of 96 barcodes that are 8 nt long, so I guess you could just reuse those same 96 on each end. But 68 out of 96 have an A or C in the first position, and a whopping 90 out of 96 have a A/C in the last position. That seems like it would be a serious problem since A/C are read by the same-color laser and Illumina says you must have color balance in your barcode combinations, even on the older machines that do four imaging cycles.