[yog77]

I would assume that you wouldn't lose many reads due to ambiguous mapping if you aligned 2x50 or even 2x75bp reads the whole genome instead of just your region of interest. It might be a bit quicker but shouldn't make such a big difference. If in doubt you could just compare the number of mapped reads against the whole genome with your region of interest, and if they don't differ very much i would possibly use the whole genome approach as this can be informative whether your experiment worked the way you intended and it is probably easier to justify for a publication at some point...

If I had a choice I would opt for 2x50 or 2x75bp reads, the latter might need to be run through a quality and/or adapter trimmer just to be sure. Low quality sequence can lead to wrong methylation calls, in rare cases even to mis-mappings (which generally produce random methylation calls). And of course many mismatches can bring down your mapping efficiency quite quickly if you use reasonably strict mapping parameters. So I suggest short to medium reads and possibly quality trimming, then you should be fine. Let me know if I can be of any further help with your project.

Thanks for the informative reply will give it some more thought and may some more questions thanks

[Jeremy37]

You should ask whoever is doing the sequencing for you what to expect in terms of read quality towards the end of the reads. When we were sequencing with the GA, we had extremely high quality all the way to base 100 -- often a median > Q35.

With the hiseqs they have been optimizing some things. We have had some runs "fail"... but they were eventually redone, and typically we have quality >Q30 at read 100. Especially since you expect to have many mismatches, I think that the value of longer reads would be very high for your application. You can always quality trim your reads using a tool like fastx (fastq_quality_trimmer). Even when the quality trails off at the end of a run, you will get a large fraction of the reads that don't need to be trimmed.

[yog77]

Thanks All for the informative answers.

Hi all I have another Question regarding this approach:

Q2) Secondly a concern some of my colleagues have highlighted is that using a PCR approach we would get over-representation of the start and end of the reads (i.e. the start and end of the desired 200bp amplicon) and a much lower if not absent coverage of the middle portion. Would anyone have any comments as to whether this would be the case and if so are there ways around this?