Jianguang, This is not recommended. The value of the paired relationships would be lost. Using an estimated Mean Inner Distance is a much better solution. This is keeping in mind that testing different values may be necessary to obtain the optimal results for any dataset. Your situation about the reported vs actual sizing is not unique and does not mean that the data is poor (when considered as a single factor). Searching an online NGS website such as seqanswers.com about the topic will being up several threads where this is discussed. Should you have outstanding concerns about this particular parameter, please consider contacting the tool authors at tophat.cufflinks@gmail.com for advice. Best, Jen Galaxy team On 8/15/12 9:59 AM, Du, Jianguang wrote:

Dear All,

I have some paired-end datasets to be analyzed, but I am not sure about their Mean Inner Distance between Mate Pairs.

Can I convert these paired-end datasets into single-end ones and use them as single-end dataset as follows?

1) Use the tool "Manipulate FASTQ" to convert the sequence of reverse reads into its reverse-complement counter part, so that all of the reverse reads actually become forward reads.

2) run Tophat on the manipulated datasets as single-end ones.

Thanks.

Jianguang

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