Sequencing Primers contain in-line barcodes

[Seq_learner]

Hi all,

I want to use dual-indexed paired-end sequencing of amplicon libraries on Miseq. My custom Read 1 and Read 2 sequencing primers have in-line barcodes for multiplexing the DNA samples. I would like to ask if I can omit the Index read step in this case?

Regards

[nucacidhunter]

Do you mean indexed PCR primers? Sequencing primers cannot be indexed.

[jdk787]

If you mean that your library inserts will contain inline barcodes (I assume at the beginning of your R1 and R2 read) that you will use to demultiplex, then yes you can omit the index read.

[Seq_learner]

Thanks jdk787. And sorry for the confusion.
Yes, I mean that I want to add the barcodes adjacent to the sample DNA and read from the same sequencing primer as part of the sequence read.

[SylvainL]

I use this technique sometimes... Do not forget to add some "N" at the beginning of your primers then, otherwise the colony detection may be inefficient (or you will have to spike PhiX at high percentage)... By experience, 5N are enough...

[Seq_learner]

Thanks Sylvainl for your comment and advice. Yes, my primers have the addition of NNNN mixed bases, but only 4 Ns.

[Vasco]

Hey Seq_learner

We are running all our illumina runs without the indexing reads (index length set to 0 prior to the run).

We additionally vary the length of our inline barcodes, which shifts all amplicons against each other, increasing sequencing diversity. Further, we switch the illumina tails for half the samples in the library (flow cel bind and sequencing primers) , so for half the samples sequencing starts from the reverse direction, further increasing diversity. We get away with using only 5% PhiX spike in with this strategy, and we don't use any N's.

See http://journals.plos.org/plosone/art...l.pone.0130324 for primer examples (Figure S1) and Figure S2 for diversity comparisons.

Best
Vasco

[liaoyunshi]

Sorry to ask a naive question, may I know how to add the inline barcodes adjacent to the sample DNA, through the specific design of PCR primer? I wonder if there is any kit can ligate the inline barcode with the adapter to the PCR products?

Thanks!

[Seq_learner]

Hi liaoyunshi,

I used PCR to added the in-line barcodes to ~ 300 bp of DNA amplicons with designed primers.

If you want to use the ligation method, you can make double-stranded adapters by annealing two indexed oligos and then ligate the adapters containing the in-line barcodes to your end-repaired DNA fragments.

There are ligation kits that you can purchase but you will need to design the in-line barcoded primers or adapters to use.

[nucacidhunter]

Sorry to ask a naive question, may I know how to add the inline barcodes adjacent to the sample DNA, through the specific design of PCR primer? I wonder if there is any kit can ligate the inline barcode with the adapter to the PCR products?

Thanks!

You can add inline barcode to the 5' end of primer(s) to generate barcoded amplicons. If the amplicon targets limited number of region resulting in low diversity then you can have variable length barcodes to increase sequence diversity. Depending on polymerase used for amplification there are two options to add adapters:
1- non-proof reading polymerase will add a non-template 3' A so you can use ligate Y adapters directly. QIAseq 1-Step Amplicon Library Kit can be used for this.
2- If proof reading polymearse is used then you can use any commercial DNA library prep kit which does end-repair, A tailing and adapter ligation replacing size-selection step (if applicable) with 1x bead clean up (for amplicons over 150 bp)

If you want to ligate barcoded adapters to non-barcoded amplicons or sheared genomic DNA then you will need to design oligos yourself and prepare adapters. This option generally will be costly and cost benefit need close attention.

[Vasco]

Sorry to ask a naive question, may I know how to add the inline barcodes adjacent to the sample DNA, through the specific design of PCR primer? I wonder if there is any kit can ligate the inline barcode with the adapter to the PCR products?

Thanks!

You can also directly order the primers already including illumina adapters, inline barcodes and amplification primers and run them in a one step or (preferably) 2 step PCR protocol (2 step = 1 st PCR with normal primers, 1ul product into 2nd step with tailed primers which include inline barcodes for tagging as well as illumina P5/P7 adapters needed for sequencing and flow cell binding.

Best
Vasco

[liaoyunshi]

Hi Seq_learner, nucacidhunter and Vasco,

Thanks for all your kindness. I do understand your protocol, but one bad news is that my DNA amplicons are much longer than several hundred bp, so I need to do fragmentation after PCR, and that makes the inline barcode included primer scheme not doable.

So I have to turn to ligation scheme, I think end-repair, A tailing and ligation enzyme are not the problem, my only concern is the generation of "artificial" adapter. It will be ligated to the DNA fragments as a double strand oligos, just like the format of adapter in commercial illumina kit. But I do not know if any oligo synthesis company can generate directly?

BTW, although I cannot use the inline barcode primers, but I think the design idea of the inline barcode is similar, so may I know how to design the sequence of the inline barcode, using any software or just refer to illumina index?

Thanks very much.

[nucacidhunter]

You can add barcodes to primers in your case if you amplify large targets with few overlapping amplicons.

For barcoded adapters you can use the Illumina adapter oligos and add the designed barcodes to your oligos and anneal. Any oligo manufacturer will do it. Following software can be used for barcode design. There might be some others as well.

http://www.deenabio.com/services/gbs-adapters

[liaoyunshi]

Hi nucacidhunter,

Yes I amplify large targets with few long overlapping amplicons and then to assemble it, but I need to do the fragmentation to make the size suitable for illumina sequencing. So if I use the barcode fusion primer, I can add the inline barcodes to the long amplicons but cannot add the inline barcodes to all the fragmented short DNA products, that make the inline invalid.

PS, for your barcoded primer, what purified level you will use? HPLC?

PPS, after sequencing, how can you de-multiplex such inline barcodes, as the inline barcode sequence should be included as the first few bases in the sequencing read.

Thanks.

[nucacidhunter]

You can have either desalting or gel purification. The trade off is between cost of purification and potentially loosing reads because of short and incomplete or oligos with deletion that will affect both PF% and quality of reads. If you have the skills you can also design half length adapters and add the rest of adapter sequences with enrichment PCR to save on costs.

Inline barcodes will be at the beginning of both read 1 and 2 if doing paired end sequencing. Some people just write a simple script to demultiplex the inline barcodes or you can use demultiplexing module of STACKS or other software used for GBS analysis.