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
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
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