How to make HiSeq indexed paired-end library with homemade oligos?

[ostrakon]

Hi guys, this is my very first post in this forum. I am new to Illumina sequencing, and I am learning a lot from this forum.

We would like to index and sequence multiple samples containing 150-bp amplicons with HiSeq paired-end reads. Because adding indexes and adapters costs $300/sample at our local facility, we would like to synthesize the oligos and prepare the library on our own. So I have requested the index and adapter sequence Letter 2011-01-11.pdf from Illumina TechSupport.

I have a few questions on making HiSeq-compatible indexed paired-end library with homemade oligos for amplicons:

1. I am not sure what oligos to use for the purpose of our experiments. Shall I use the oligos from the Multiplexing Sample Prep Oligo Only Kit, the Paired End DNA oligo squences, or the TruSeq RNA and DNA Sample Prep Kit?

2. Although I assume we should use the oligos from the TruSeq Kit, I still hope to clarify the bewilderment I have on the multiplex PCR. Thanks to the illustrations posted by members on this forum, I understood the formation of Y structures using the PE adapters, and how the PCR works to generate a symmetric PCR product using the PE PCR primers 1.0 and 2.0. But what are the PCR primers for indexes 1-12 for in the Multiplexing Sample Prep Kit? So do we need to include three primers in the PCR to generate indexed PCR products? How does it work? I couldn't find the protocol for it, either. So could someone help me by providing some explanations or illustrations on how the multiplex PCR works?

3. If we need to use the oligos from the TruSeq Kit, shall we use these adapters instead of the ones from the Multiplexing Sample Prep Kit? Is the TruSeq universal adapter used as one adapter, and one of the TruSeq Indexed Adapters as the other adapter during the ligation? So what primers shall we use in the PCR? I noticed that the TruSeq universal adapter is the same as the PE PCR primer 1.0.

4. If we need to use the oligos from the TruSeq Kit, can we use what we have in the lab instead of buying everything mentioned in the kit? For example, is the Ampure XP kit better over Qiagen PCR purification kit? Can we stain the gel with EB rather than Sybr Gold? What enzymes are recommended for end repair, adenylation, ligation, PCR, and etc? Could someone share a “homebrew” protocol using alternative reagents and kits that worked for you? Or, is it recommended that we only synthesize our oligos, but still buy the TruSeq kit?

5. In Quail et al. 2008, Nature Methods 5(12):1005-1010, they proposed direct sequencing of short amplicons in the supplementary protocol 10. Would this work on HiSeq?
Alternatively, Since we start from amplicons to make the library, can we actually add all these adapter and index sequences by incoporating them into really long primers and generate the amplicons directly?

Well, this post is loaded with so many questions, but I am really puzzled on what oligos to use as adapters and PCR primers, how the PCR works for making HiSeq-compatible indexed paired-end library, and how to actually do it.

Thanks a lot for any suggestions or experiences!

-ostrakon

[Corey]

Re question 5, we have had good outcomes routinely using 96 different indexes added directly to our primers, see here for an early version of the protocol.: Genome Res. 2009 Oct;19(10):1836-42. Epub 2009 Jul 21. Quantitative phenotyping via deep barcode sequencing.
Good luck, Corey

[ostrakon]

Thanks for your reply, Corey. Your paper inspired me greatly.

So I have made a draft graph of the PCRs I want to do with the amplicons. Would you guys think it will work or not? Green and yellow represent the adapters.

I put it in my picasa album:

[greigite]

Re question 4, we get higher yields from Ampure XP purification than from Qiagen (or other brands) of column- typically with Ampure we lose ~25% of material per purification step while with columns it can be ~50%. We used the Pippin prep instrument instead of gel extraction to size select the library but if you are using gels, it might be better to use a stain like GelRed that doesn't require UV light. Before TruSeq kits came out we used the NEB library prep kit with custom adapters and it worked fine, except that our inline barcodes gave crappy results on the HiSeq.

[pmiguel]

Hi Ostrakon,
You might want to just use the TruSeq kit. The new ones only run ~$50 in reagents per library. You could probably get the price/library much lower by whipping up your own set of reactions and sourcing all the oligos/reagents separately. But, realistically, chances are you are going to spend a lot of time perfecting methodologies already folded into the TruSeq kit.
So if you are going to be making thousands of libraries, it may be worth it to put in the extra time to get everything working using a much cheaper set of reagents. But for just making a handful--not worth the effort.
--
Phillip

[HESmith]

Hi Ostrakon,

Your PCR will not work as diagrammed. Your green primers (shown in first cycle PCR) will anneal to the same sites as your yellow primers (second cycle), so you'd end up with all possible combinations of green/yellow, 5'/3' ends. I'll leave it to you to sort out what happens in subsequent rounds of amplification.

Harold

[JLY]

Re question 4, we get higher yields from Ampure XP purification than from Qiagen (or other brands) of column- typically with Ampure we lose ~25% of material per purification step while with columns it can be ~50%. We used the Pippin prep instrument instead of gel extraction to size select the library but if you are using gels, it might be better to use a stain like GelRed that doesn't require UV light. Before TruSeq kits came out we used the NEB library prep kit with custom adapters and it worked fine, except that our inline barcodes gave crappy results on the HiSeq.

Hi greigite,

I'm new to this forum and happened to be looking through this thread for information re illumina PE multiplex sequencing using the HiSeq system when I saw your post. I've designed custom PE adapters with an in-line TruSeq illumina index sequence at the 3' end of upstream adapter before the last 3' nucleotide (complementary sequence inserted at the 5' end of the downstream adapter).

Originally I was going to use the GAIIx system. I'm now looking at using HiSeq as it should work out faster, and I can pool more samples per lane without loosing reads, making it cheaper.

My concern is that my adapters and PCR amplification primers may not be suitable for HiSeq. Also, the protocol I use for PE sample preparation has been tried and tested with the GAIIx but not HiSeq.


Jo