@kmcarr: I will concede that the constraint is mostly, if not entirely, theoretical since the adapter sequencer should be known -- certainly it will be by us service providers and this information should be passed onto our customers. A 'adapter-knowledge-free' program would only be useful in extremely rare cases or as part of a thought experiment.
I had not considered Trimmomatic's Palindrome mode since I never use that part of Trimmomatic. Thanks for the tip.
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Originally posted by westerman View PostAs a followup, it turns out that the samples in question did not (for the most part) look like the 2nd example I gave -- i.e., with the desired fragment fully contained in R1 and R2 with R1 starting inside R2 and vice-versa. Instead most of the reads looked like the 1st example thus we could use normal Panda/Flash methodology on them.
It might still be interesting to develop an 'adapater-knowledge-free' stitching/merging program. But that is a task for another day.
If you have a priori knowledge of the adapter sequences then Trimmomatic, using it Palindrome trimming mode, handles cases like #2, but not in exactly the way you asked about. I makes not attempt to "merge" the two reads. It simply clips the adapter from read 1 and discards read 2 entirely as it contains no additional data beyond that which is contained in read 1.
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As a followup, it turns out that the samples in question did not (for the most part) look like the 2nd example I gave -- i.e., with the desired fragment fully contained in R1 and R2 with R1 starting inside R2 and vice-versa. Instead most of the reads looked like the 1st example thus we could use normal Panda/Flash methodology on them.
It might still be interesting to develop an 'adapater-knowledge-free' stitching/merging program. But that is a task for another day.
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Originally posted by mcnelson.phd View Post... but the new version of Reporter incorporates a read "Stitching" feature that might do exactly what you want.
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Originally posted by GenoMax View PostRick,
It sounds like you do not want to trim (adapters) before the merge, is that a requirement?
Indeed, the longer lengths are making for interesting possibilities.
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It's probably too late right now if your run is already doing, but the new version of Reporter incorporates a read "Stitching" feature that might do exactly what you want. You'll have to manually add the flag to your sample sheet and reprocess your data if you want to try it. Check out the full guide on Reporter for what the actual flag is and what options are associated with it.
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This group published along these lines:
Backgound High throughput sequencing is beginning to make a transformative impact in the area of viral evolution. Deep sequencing has the potential to reveal the mutant spectrum within a viral sample at high resolution, thus enabling the close examination of viral mutational dynamics both within- and between-hosts. The challenge however, is to accurately model the errors in the sequencing data and differentiate real viral mutations, particularly those that exist at low frequencies, from sequencing errors. Results We demonstrate that overlapping read pairs (ORP) -- generated by combining short fragment sequencing libraries and longer sequencing reads -- significantly reduce sequencing error rates and improve rare variant detection accuracy. Using this sequencing protocol and an error model optimized for variant detection, we are able to capture a large number of genetic mutations present within a viral population at ultra-low frequency levels (
They align the raw reads and analyze that rather than merging. There is a second paper that came out more recently as well, but I can't dredge it up. My lab should have our version out soon, too. Gary Schroth at Illumina said he was pushing long ago to have this the standard output of the Illumina machines as a way to get separation on error rate with other platforms, so it is funny that years later there is a sudden wave of labs all independently coming up with the idea.
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I use SeqPrep for exactly that purpose, although I do an extra careful adapter stripping before and after merging to clean up the errors. It did an ok job without the extra step, but I wanted the reads as error-free as possible. I can reliably find alleles at the 0.03% range by doing that.
I look at the length of the merged reads and trim back if they are a size range where partial adapters would have been present. But your approach would work too, I think.Last edited by SNPsaurus; 08-27-2013, 09:10 AM.
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@GenoMax: Your idea should work but doing it for an entire miSeq run sounds like a long processing time. I was hoping for a quicker and one-stop solution.
@McNelson.phd: No, I haven't tried SeqPrep but from my reading of it -- and your description -- it sounds like it would act the same as Panda and Flash: not good for when there is no prior knowledge of the adapter. I will install it though and give it a spin.
Real data coming off the sequencer later today!
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Have you tried SeqPrep?
I know I've tried it on Nextera data and by giving it the Nextera adapter sequence it was able to spit out reads with 100% overlap but whose length was < 250bp, which would fit what you're talking about. What I can't say is how it would handle the "adapter" sequences that might hang off the ends if you don't provide it with any sequence to look for.
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A pair-wise aligner (that can export a consensus, followed by an appropriate trim) should work right?
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As Phillip SanMiguel said to me in private email and which may clarify my post:
So the reads may all have a few (1-5 bases) of adapter at the their 3' ends. A better way to trim them would be to compare R1 and R2 -- the first base of each should point out the last base of the the other. If PANDA had a setting to remove single stranded sequence from pair merges, that would be good.
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Merger/overlapper for fully contained fragment
I am trying to find a tool that would do merging/overlapping of PE reads when the fragment is fully contained within the reads and without having to know the adapters ahead of time. The program PANDA and FLASH (and others) will merge PE reads into a single read however they are geared towards cases where the fragment is a subset of the read. E.g.
Code:R1: ----------> R2: <---------- Frag: ----------------
Code:R1: -------------> R2: <-------------- Frag: ------------
Hope that this makes sense. Any suggestions? Thanks.Tags: None
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