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Old 05-25-2015, 02:06 AM   #1
aaron_tao
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Default Would it be OK if the Read 1 and Read 2 Seq Primer are identical for Myseq?

Dear Friends,

I am designing a pipeline to construct a 2X250bp Pair End lib for Myseq.
I have identical adapter on both end of my DNA. To avoid the waste of sequencing bases, Would it be OK if the Read 1 and Read 2 Seq Primer are identical?

Best
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Old 05-25-2015, 02:52 AM   #2
mastal
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You would have to use the reverse complement of the sequence for Read2.
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Old 05-25-2015, 03:49 AM   #3
nucacidhunter
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Quote:
Originally Posted by aaron_tao View Post
Dear Friends,

I am designing a pipeline to construct a 2X250bp Pair End lib for Myseq.
I have identical adapter on both end of my DNA. To avoid the waste of sequencing bases, Would it be OK if the Read 1 and Read 2 Seq Primer are identical?

Best
Identical adapters in both ends will not work because:
1- different adapter sequences are required for clustering and bridge amplification.
2- even if somehow you mange to cluster, during sequencing read1 and read 2 primers are dispensed from different tubes in cartridge and bind to related motif on adapter to prime sequencing. If you library fragments are flanked by P7 or P5 adapters then one of the read primers will not bind to related read primer binding site.

It is not clear what you mean by "avoid waste of sequencing bases".
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Old 05-25-2015, 03:58 AM   #4
aaron_tao
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Quote:
Originally Posted by mastal View Post
You would have to use the reverse complement of the sequence for Read2.
Thanks for your reply. But I think for Read 2 Seq primer, is the same as Read 1 Seq primer as show in the figure I attached. How do you think?

seq.PNG
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Old 05-25-2015, 04:08 AM   #5
aaron_tao
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Quote:
Originally Posted by nucacidhunter View Post
Identical adapters in both ends will not work because:
1- different adapter sequences are required for clustering and bridge amplification.
2- even if somehow you mange to cluster, during sequencing read1 and read 2 primers are dispensed from different tubes in cartridge and bind to related motif on adapter to prime sequencing. If you library fragments are flanked by P7 or P5 adapters then one of the read primers will not bind to related read primer binding site.

It is not clear what you mean by "avoid waste of sequencing bases".
Thanks for your reply. I would use P7/5 adapter in addition to my adapter.
For "avoid waste of sequencing bases", I mean if I use the official seq primer, I would have to seq the adapter constant region for all the reads.
Instead, I want to use this constant region as seq primer. But the primer would be the same for both Read1 and Read2. How do you think?
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Old 05-25-2015, 04:44 AM   #6
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It seems that you want to amplify target of interest using PCR with region specific primers fused to partial Illumine adapter sequences. In tat case you will need custom sequencing primers. If this is the case you would find published work and also threads in this forum.
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Old 05-26-2015, 05:57 AM   #7
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If both reads have the same primer, then you will get both reads at the same time, which means you will be reading different bases at the same time from each cluster, which means you will get no usable data.
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Old 05-26-2015, 12:32 PM   #8
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I believe for Nextera adapters the sequencing primers are indeed identical for both forward and reverse reads. This works because of the re-clustering happening between these reads.
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Old 05-26-2015, 08:50 PM   #9
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Nextera converts the very short adapters left by the transposase into different adapters with long PCR primers. Fragments with the same ends are competed out by suppression PCR.
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Old 05-26-2015, 10:35 PM   #10
aaron_tao
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Quote:
Originally Posted by nucacidhunter View Post
It seems that you want to amplify target of interest using PCR with region specific primers fused to partial Illumine adapter sequences. In tat case you will need custom sequencing primers. If this is the case you would find published work and also threads in this forum.
You got it. The problem is between the target region and illumina adapter sequence, I have another adaptor on both ends, illustrated bellow:

p5--ATCGGCCC--targetofinterest--GGGCCGAT--P7
P5'--TAGCCGGG--targetofinterest--CCCGGCTA--P7'


Do you think Could I use "ATCGGCCC" as both Read1&Read2 Seq Primer?

Thanks.

Last edited by aaron_tao; 05-27-2015 at 02:46 AM.
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Old 05-27-2015, 03:41 AM   #11
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Only if "targetofinterest" is always a palindrome.
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Old 05-27-2015, 04:06 AM   #12
mastal
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Quote:
Originally Posted by aaron_tao View Post
You got it. The problem is between the target region and illumina adapter sequence, I have another adaptor on both ends, illustrated bellow:

p5--ATCGGCCC--targetofinterest--GGGCCGAT--P7
P5'--TAGCCGGG--targetofinterest--CCCGGCTA--P7'


Do you think Could I use "ATCGGCCC" as both Read1&Read2 Seq Primer?

Thanks.
You won't get properly paired reads.

When you produce Read1 the primer will hybridize to clusters with both orientations, and
you will get reads from both orientations as Read1.

Then when you do Read2, the clusters with one of the orientations will have been removed, and you will get reads from only one of the orientations.


You can do single-end reads instead of paired-end reads.
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Old 05-27-2015, 04:17 AM   #13
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But the sequencer doesn't know which end you want to be read 1 vs. read 2, if you give it the same primer for both, so it will read both ends at the same time in every cluster, and every cycle with give you a mix of two different bases (from opposite ends of the insert), resulting in no useful base calls.

Let's take a step back. What is the purpose of this question in the first place?

Quote:
Originally Posted by aaron_tao View Post
To avoid the waste of sequencing bases
Even if this scheme were possible, how would it accomplish that? I think we're struggling to solve an ill-posed problem.
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Old 05-27-2015, 05:17 AM   #14
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Some of the responders need to review the Illumina technology. Only one of the two strands is retained after clustering (the second is cleaved), so it's impossible to read both ends of a cluster simultaneously. And the original poster is correct in that the adapter sequences immediately adjacent to the insert are identical on both sides (true for both Nextera and TruSeq libraries). However, the annealing temperature of the suggested primer (ATCGGCCC) is too low; see Illumina recommendations for custom primer design and loading on the MiSeq.
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Old 05-27-2015, 06:19 AM   #15
Jessica_L
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Quote:
Originally Posted by aaron_tao View Post
You got it. The problem is between the target region and illumina adapter sequence, I have another adaptor on both ends, illustrated bellow:

p5--ATCGGCCC--targetofinterest--GGGCCGAT--P7
P5'--TAGCCGGG--targetofinterest--CCCGGCTA--P7'


Do you think Could I use "ATCGGCCC" as both Read1&Read2 Seq Primer?

Thanks.
I believe HESmith is correct. After the linearization, blocking and denaturation steps, there would only be one TAGCCGGG sequence available for the ATCGGCCC primer to anneal to. The hardest part of doing this kind of thing, IME, is to make sure all adapters and primer sequences are in the correct orientation (and reverse complemented if need be).

It actually makes a bit of sense to try and move the sequencing primer toward the 3' end of the adapter region since the first 8 bases of every sequence read would be 'ATCGGCCC'. To my thinking, it's less about having 8 bp of adapter sequence at the front of each read (wasting sequence data) and more about increasing base pair diversity early on-- I know that Illumina claims newer instruments are supposed to have an easier time with low complexity libraries through the first 25 bases, but it's still just a red flag for me (and admittedly, I haven't run anything like this in a while).


In the case of the too low Tm, I think there are two possibilities to solve that (hopefully the OP hasn't ordered any oligos yet):

1) The 5' end of the primer needs to be extended into the illumina adapter. The downside of that is that you lose the identity of the primers between both reads (i.e. one primer has P5 at its 5' end, the other would need P7).

2) But why not just make the original primer/adapter sequence a little longer? You can retain the identity between sequence primers for both read 1 and read 2, and extending out from the 5' end won't alter the annealing at the 3' end.

I guess my follow-up question to the OP is whether there's anything unusual about the target of interest-- are the fragments already unusually long or do the thermodynamic properties present any particular difficulties? In other words, is there something special about that 8bp sequence or are there reasons not to make it longer?
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Old 05-27-2015, 02:47 PM   #16
jwfoley
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I misunderstood the question. Yes, as long each molecule's outer sequences are still the two distinct P5 and P7 adapters, not identical, the innermost sequence can match. (The innermost dozen bases match on the original Solexa adapters too, not just Nextera, because that's the complementary portion of the Y-linker.)

But because of the Tm issue, it'll need to be a longer primer than that. On the other hand, because the inner sequences of the Illumina adapters match, it might still be the same primer for both ends.

Don't try this unless you really know what you're doing.
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Old 05-29-2015, 10:05 AM   #17
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This can probably work, as long as you have the P5 and P7 sequences surrounding the sequencing primer. That is, it's not exactly an inverted repeat.

The only issue you will have is competition for hybridization from the reverse complement that exists in cis on the library that's on the flow cell versus your incoming sequencing primer. But if you flood it in at 0.5uM you should still be able to get a large number of clusters.
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Old 06-03-2015, 08:08 AM   #18
aaron_tao
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Thanks so much. It help me a lot. I would design a longer adapter according illumina's guideline.
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