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  • Retrieving single and reads from paired end read sequencing run

    Hi everyone,
    As a first single cell RNAseq experiment, we would like to compare between single end and paired end sequencing. So at the beggining we wanted to perform the two sequencing on the same library. But our facility told us we should only run a paired end seq and then we could retrieve the single end reads from the paired end read sequening. My question is, is running a single end sequencing the same as running the PE seq and then just take into account only read 1?
    Thanks for your help

  • #2
    Yes, on Illumina systems paired-end is basically an optional extension of single-end sequencing. Lets say you have a Nextseq platform and a kit for 150 cycles (means reagents to make 150 basecalls per cluster), then you have the choice to go either 150bp into the fragment (single-end) or 2x75bp (paired-end, forward-and reverse read). The sequencing data are stored in so called fastq files, which contain the called bases and the quality information. In case you have single-end data, you get one file per sequencing index, in case of paired-end, you get two files. One for the forward, one for the reverse read. Usually, the files are then labelled like name_1.fastq (fwd-read) and name_2.fastq (rev-read), or similar. Hope that helps.

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    • #3
      Thanks for your answer. Just for precision, we ran 2*50bp sequencing and we wanted to copare with 1*50bp sequencing.
      Cheers

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      • #4
        You are sampling the same fragment from two ends. Since these are 50 bp reads they would not be expected to overlap. You could treat the two reads as independent single end reads but when you map them only reads that map concordantly (at expected distance of the fragment size) would be "correct" pairs (unless you are expecting translocations etc for some).

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