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Old 11-06-2017, 12:51 PM   #1
kbojones
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Default ddRADSeq Libraries

We seem to be having some difficulties creating reproducible ddRADSeq libraries.

We are noticing that after running the first of the processing steps (process radtags) - which removes the barcodes, filters for the rad sites, and checks for sequence quality - we have some evidence of perhaps incomplete restriction digestion.
For instance - we have:
rad cut site (i.e, CATG from NlaIII) - followed by target sequence - followed by the second cut site (i.e., MluCI - AATT) - followed by more sequence.

Shouldn't the sequence end at the second cut site? Has anyone tried a sequential digest? We have been running the test libraries at 150bp SE on a MiSeq.

Any help is greatly appreciated.
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Old 11-06-2017, 03:32 PM   #2
nucacidhunter
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A single digest is far better than sequential digest if both enzymes are active in the same temperature and buffer (yours are).

In ideal reaction condition incomplete digestion should be minimal. I wonder if the sequences beyond MluCI site are untrimmed adapter sequences.
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Old 11-08-2017, 06:50 AM   #3
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Thanks for the reply nucacidhunter. After looking over the data again there is a minimal amount of adapter attached after the cut site. The majority of reads are partial digestion by the MluCI restriction enzyme. I'm not sure why this is. We are starting with 200 ng of gDNA and adding 10 units total (1 ul) of each enzyme to a 50 ul reaction. It is then incubating in a thermalcycler for 3 hours at 37C.

I'm wondering if we should try adding more of the MluCI RE compared to the NlaIII RE or extending the digest to 6 hours.
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Old 11-08-2017, 10:47 AM   #4
SNPsaurus
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Could you be getting chimeras of adapter - NlaIII - fragment 1 - MluCI - fragment 2 - MluCI - adapter?

You would see different sequences after the first MluCI for a given locus if so.

These are both 4-cutter enzymes... are you trying to sample an enormous number of loci?
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Old 11-08-2017, 01:31 PM   #5
nucacidhunter
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You can add more RE provided that the final combined volume does not exceed 10%. It is better to do a test digest on MluCI to rule out bad batch of RE.

The issue mentioned by SNPsaurus could be result of fragments ligating to each other rather than to adapters which could be overcome by increasing adapter quantity in reaction. It also can result from overcycling.

Last edited by nucacidhunter; 11-08-2017 at 01:47 PM. Reason: Omitting unnecessary line.
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Old 11-08-2017, 02:46 PM   #6
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I was thinking about this some more, and I think the use of two 4-cutters may be creating conditions that enrich for this sort of artifact.

What is your size selection range? Most people do ddRAD with 300 bp fragments for PE 2x150 reads. If that is the case, you will have very few fragments available at 300 bp, since the genome is being chopped up into 125 bp fragments by the use of two 4-cutters. Even if chimeras happen at a low rate, these chimeras will be now long enough to be in the size selection range, and could be present at that size range as an appreciable fraction compared to the unlikely desired NlaIII-MluCI fragment of that size.
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Old 11-09-2017, 08:14 AM   #7
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Wow. Thanks so much for the insight. Right now we are supposedly adding 5 fold adapter concentrations but we could up that. The calculations are based off of the enzyme trial step prior to the protocol, which you would think also has this incomplete cutting.

Would using a 6-cutter and a 4-cutter enzyme in the double digest be more advantageous? Perhaps SphI (6 cutter) and NlaII (4 cutter).

We were also thinking about selecting a larger range of basepairs in size selection step. Last round we did 300-400bp on our Pippen Prep. This time we were planning on doing 400-600bp, since this will be SE sequencing.

We will be receiving a positive control from NEB, to test our enzyme function. Our current project is using deep-sea fish genomic DNA. There is a slight chance that the problem could be with this kind of genomic DNA.
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Old 11-09-2017, 10:42 AM   #8
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I'm curious what your goals are for the project. There might be several million of each site in the genome (if it is 1 Gb). Even if you took the largest 5% of fragments, that could be several hundred thousand loci being amplified and sequenced. Getting good read depth on that number of loci will require lots of sequencing per individual (10M reads or more). Plus the artifacts from SNPs creating new 4-cutter sites in your fragments would mean a huge rate of missing data in any kind of genetically diverse population.

If you are just looking at population structure, then I would pair up a 6-cutter or 8-cutter with a 4-cutter. You could estimate the number of 6 or 8-cutter sites in the genome, then figure out from your size selection what portion would be kept (so keeping 400-600 bp fragments will mean keeping fragments bigger than the expected ~250 bp (the usual rate of a 4-cutter cutting)... only 10% of fragments will be 500 bp, for example.

How many samples do you have? What's your budget for sequencing? I'd take the # loci x read depth desired and multiply by 3 or more to get how much sequencing will be needed. Loci amplify at different rates, and samples vary in how many reads they get so you need to pack in some buffer to get good depth at most loci in most samples.
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Old 11-09-2017, 12:16 PM   #9
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Using a 4 and 6 cutter combination and 100 bp window cut should give over 10k tags. I would suggest to use 5 ng of digested-ligated DNA in 10-12 PCR cycle and check the size distribution. The profile should give hints on size selection window and also can help to avoid repeat regions tags.
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Old 11-10-2017, 09:28 AM   #10
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Thank you nucacidhunter and SNPsaurus! We are going to use all of this information to remake the libraries and run another test run on the MiSeq. I will follow-up with results. Thanks again you have been more thank helpful!
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