18 lines
2.3 KiB
Markdown
18 lines
2.3 KiB
Markdown
## Paired-end / mate-pair
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http://gatkforums.broadinstitute.org/gatk/discussion/6327/paired-end-mate-pair
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<p>In paired-end sequencing, the library preparation yields a set of fragments, and the machine sequences each fragment from both ends; for example if you have a 300bp contiguous fragment, the machine will sequence e.g. bases 1-75 (forward direction) and bases 225-300 (reverse direction) of the fragment. </p>
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<p>In mate-pair sequencing, the library preparation yields two fragments that are distal to each other in the genome and in the opposite in orientation to that of a mate-paired fragment.</p>
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<p>The three read orientation categories are forward reverse (FR), reverse forward (RF), and reverse-reverse/forward-forward (TANDEM). In general, paired-end reads tend to be in a FR orientation, have relatively small inserts (~300 - 500 bp), and are particularly useful for the sequencing of fragments that contain short repeat regions. Mate-pair fragments are generally in a RF conformation, contain larger inserts (~3 kb), and enable sequence coverage of genomic regions containing large structural rearrangements. Tandem reads can result from inversions and rearrangements during library preparation. </p>
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<p>Here is a more illustrative example:</p>
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<p><strong>FR:</strong> 5' --F--> <--R-- 5' (in slang called "innie" because they point inward)</p>
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<p><strong>RF:</strong> <--R-- 5' 5' --F--> (in slang called "outie" because they point outward)</p>
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<p><strong>TANDEM:</strong> 5' --F--> 5' --F--> or <--R-- 5' <--R-- 5'</p>
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<p>The figure below illustrates this graphically along with the SAM flags that correspond to the FR and RF configurations.</p>
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<img src="https://us.v-cdn.net/5019796/uploads/FileUpload/e3/c9e87118d6e8c4b2a4e014d97a1b22.png" />
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<p>For detailed explanations of library construction strategies (for Illumina sequencers) and how read orientations are determined, please see:</p>
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<ul>
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<li><a href="http://www.illumina.com/technology/next-generation-sequencing/paired-end-sequencing_assay.html">Illumina paired-end sequencing documentation (webpage)</a></li>
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<li><a href="http://www.illumina.com/documents/products/technotes/technote_nextera_matepair_data_processing.pdf">Illumina Nextera mate-pair processing documentation (pdf)</a></li>
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