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what is a paired-end read?

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  • #16
    no not yet. i'm going to have run through with them as soon as i can. thanks for the info

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    • #17
      Hi all,
      I am still confused with the difference between pair ends and mate pairs. Is it just that you call mates a pair of sequences (ends) from the same molecule?

      Tnx

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      • #18
        Originally posted by polivares View Post
        Hi all,
        I am still confused with the difference between pair ends and mate pairs. Is it just that you call mates a pair of sequences (ends) from the same molecule?

        Tnx
        Hey...Nice last name!

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        • #19
          Originally posted by ECO View Post
          Hey...Nice last name!
          That means?

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          • #20
            Originally posted by polivares View Post
            That means?
            Olivares is my surname as well. Haven't met many before.

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            • #21
              paired-end and mate pairs

              Paired-end and mate pairs. These two denominations refer to slightly different library preparations... correct?

              Do we usually have the choice between the two? What are the practical differences between them? Anyone compared possible biases for example?

              Best,

              Yvan

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              • #22
                Illumina refers to "paired end" as the original library preparation method they use, where you sequence each end of the same molecule. Because of the way the cluster generation technology works, it is limited to an inter-pair distance of ~300bp ( 200-600bp).

                Illumina refers to "mate pairs" as sequences derived from their newer library prep method which is designed to provide paired sequences separated by a greater distance (between about 2 and 10kb). This method still actually only sequences the ends of ~400bp molecules, but this template is derived from both ends of a 2-10kb fragment that has had the middle section cut out and the 'internal' ends ligated in the middle. Basically, you take your 2-10kb random fragments, biotinylate the end, circularise them, shear the circles to ~400bp, capture biotinylated molecules, and then sequence those (they go into what is essentially a standard 'paired end' sample prep procedure).

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                • #23
                  Will original insert size of library be in SAM file header for Mate-paired sequences?

                  Would the original insert size for the library be listed in any resulting sequence alignment files (SAM/BAM for instance) for mate-paired reads (typicaly 2-5 kBp) or would it list the insert size of the paired-end reads (Typically ~500 bp). This would be important for the downstream Structural Variant analysis I can imagine...

                  I know that SAM/BAM is not (yet) produced by Illumina or Solid systems natively, but do any translators take care of this?

                  Thanks!
                  Thon
                  __________________________________
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                  Director of Product Management, Software
                  Strand Life Sciences
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                  • #24
                    Originally posted by thondeboer View Post
                    Would the original insert size for the library be listed in any resulting sequence alignment files (SAM/BAM for instance) for mate-paired reads (typicaly 2-5 kBp) or would it list the insert size of the paired-end reads (Typically ~500 bp). This would be important for the downstream Structural Variant analysis I can imagine...

                    I know that SAM/BAM is not (yet) produced by Illumina or Solid systems natively, but do any translators take care of this?

                    Thanks!
                    Take a look at the SAM specifcation. The insert size of an alignment (ISIZE) is defined as the distance between the 5' ends of the reads after mapping. The PI field in the RG tag in the header allows reads with the same expected insert size to be identified.

                    So to answer your question, if a read has its two ends 2-5Kbp or 500bp apart, ISIZE should be set accordingly. Optionally, the predicted size can be inferred from PI/RG in the header (when available).

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                    • #25
                      I think paired end refers to sequencing from the ENDS of a DNA fragment. If you're doing 75-bp paired end read using an insert size of 300bp, then the machine will sequence 1-75 and 300-225 (for simplicity, omitting the adapters)

                      Mate pair requires a completely different protocol and is typically over longer distances such as 2-5kb. If you want to sequence a 5kb mate pair library, then 5kb fragments of DNA are isolated on the gel, the ends are biotinylated, the fragment is circularized and sheared. So now when you select using streptavidin, you'll get the fragment that has the ENDS of the original 5kb fragment. This fragment is then sequenced.

                      Mate pair is more relevant in genome assembly, especially for covering repetitive sequences. Paired end can be used for anything - RNA, DNA.

                      You can get more information on the Illumina site:
                      http://www.illumina.com/technology/p...ing_assay.ilmn

                      http://www.illumina.com/technology/m...ing_assay.ilmn

                      I'm not sure how 454 or other methods define these terms

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                      • #26
                        Mate pair can also cover longer distances & is therefore more sensitive for detecting rearrangements / structural variation.

                        A third entry in this general category are what Pacific Biosciences calls strobe reads and Helicos calls dark fill. Essentially what happens is that sequence is read then there is a gap of probabilistic length then more sequence is read. This can be repeated many times to give a series of sequence islands separated by gaps with a constrained length distribution.

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                        • #27
                          I am not very familiar with all the biotechnology, so want to ask:
                          Is it possible to resolve which pair of reads come from the one, unique molecule ? (For instance, that the sequencer tracks this information somehow.) I mean usually there are a lot of reads and in diploid systems it might happen there are two 'versions' of the reads.

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                          • #28
                            I understand that a normal read pair can be aligned with either F-B or B-F orientation. What I'm not too sure about the importance of read1 and read2, does it mean anything if the mapped position of read1 is greater than read2?

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                            • #29
                              Originally posted by hege View Post
                              I understand that a normal read pair can be aligned with either F-B or B-F orientation. What I'm not too sure about the importance of read1 and read2, does it mean anything if the mapped position of read1 is greater than read2?
                              Assuming the alignment is correct that may indicate a structural variation happened at that location (you want to check what other alignments on that location tell you).
                              -drd

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                              • #30
                                Originally posted by plichel View Post
                                I am not very familiar with all the biotechnology, so want to ask:
                                Is it possible to resolve which pair of reads come from the one, unique molecule ? (For instance, that the sequencer tracks this information somehow.)
                                Yes. In any of these schemes the sequencer keeps track of the physical location in the flowcell of the first read for each polony (spot of DNA) and then

                                For ligation technologies (Polonator & SOLiD), the reverse reads are really the same technology. For Illumina, there are some clever molecular acrobatics to replace the originally sequenced molecule with it's reverse complement to do paired end sequencing. For mate pairs, I believe they just strip the old extended primer & anneal a new primer. Helicos proposed just adding unlabeled bases for a time period (hence the term "dark fill") and PacBio shuts off their laser and keeps extending (hence "strobe sequencing" is an apropos term).

                                Complete Genomics used a 4-part read structure & may have gone to even higher number of mates.

                                Keeping track of the location of polonies or individual DNA molecules is one of the core tricks to all these technologies.

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