Hola Diego,
The typical directional libraries are a result of the following procedure: the DNA is sheared and blunt-ended and the Illumina adapters are ligated onto the reads. This is done in a fashion so that a specific adapter sequence binds to the 5' end of the reads (from both the forward and reverse strand), and a different primer binds to the 3' ends. This works because the first part of the adapter is initially complementary but they then fork so that they are no longer complementary. It is exactly this forking part of the adapter that will only allow the former top and bottom strand reads that have the adapter on their 5' end to anneal to the primers on the flow cell - and these get sequenced as Read 1 (of paired end files) or as the only read of single-end sequencing.
Amplicon sequencing is indeed different because this normally works by bisulfite converting and then amplifying a specific sequence. Because this is usually done for the top strand only I would expect your sequences to align to either the OT or CTOT strands, but this depends a bit on the kit design. I am happy to advise on that if you could send me the mapping reports of your --non_directional run.
And to your last point: No, single-end and paired-end reads should not be combined but run individually. (Having said that the reduplication and methylation extraction steps should detect automatically which kind of file they are working with, but if I were you I would still keep them separate to be safe). If you have sequenced the same sample several times with SE and PE reads you could merge the results e.g. at the bedGraph conversion stage once you have deduplicated and extracted the files individually.
Hope this helps getting you started, Cheers, Felix
The typical directional libraries are a result of the following procedure: the DNA is sheared and blunt-ended and the Illumina adapters are ligated onto the reads. This is done in a fashion so that a specific adapter sequence binds to the 5' end of the reads (from both the forward and reverse strand), and a different primer binds to the 3' ends. This works because the first part of the adapter is initially complementary but they then fork so that they are no longer complementary. It is exactly this forking part of the adapter that will only allow the former top and bottom strand reads that have the adapter on their 5' end to anneal to the primers on the flow cell - and these get sequenced as Read 1 (of paired end files) or as the only read of single-end sequencing.
Amplicon sequencing is indeed different because this normally works by bisulfite converting and then amplifying a specific sequence. Because this is usually done for the top strand only I would expect your sequences to align to either the OT or CTOT strands, but this depends a bit on the kit design. I am happy to advise on that if you could send me the mapping reports of your --non_directional run.
And to your last point: No, single-end and paired-end reads should not be combined but run individually. (Having said that the reduplication and methylation extraction steps should detect automatically which kind of file they are working with, but if I were you I would still keep them separate to be safe). If you have sequenced the same sample several times with SE and PE reads you could merge the results e.g. at the bedGraph conversion stage once you have deduplicated and extracted the files individually.
Hope this helps getting you started, Cheers, Felix
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