<p>Hi all,
here are some questions about <strong>paired-end sequencing</strong> for NGS:</p>

<ul>
<li>What are the main differences between <strong>mate-paired</strong> sequencing and <strong>paired-end</strong> sequencing; Should I care when I use tools like 'samtools', maq, etc.... ? Should one, and only one short read, should be paired with another one (1-1)?</li>
<li>What is <strong>removing duplicates</strong> ? does it mean that a <strong>pair</strong> of short reads has been mapped at two distint positions on the genome or does it mean that a pair matched too many time at one position ?</li>
<li>Knowing that <strong>bwa sampe</strong>  "Generates alignments in the <a href='http://samtools.sourceforge.net/SAM1.pdf'>SAM</a> format given paired-end reads. Repetitive  read pairs will be placed randomly", is there any need to "remove the duplicates" ?</li>
<li>How does picard <strong>MarkDuplicates</strong> work ? How can I find the reads that have been 'tagged' ? will it remove the reads from the <a href='http://samtools.sourceforge.net/SAM1.pdf'>BAM</a> file ?</li>
</ul>

<p>Thanks</p>

<p>Pierre</p>

<p>Hm.  Old questions, so nobody will read this, but I'm not entirely happy.  Here are <em>my</em> answers:</p>

<ol>
<li>Paired ends is supported by some technologies (Illumina and Sanger), where it is possible to sequence from both ends of a clone.  Mate pairs involves making circular fragments using a linker sequence, and fragmenting them around the linker, and then sequencing the result.  Illumina will read from each end of the fragment, 454 (and I believe Solid?) will read through it all.</li>
</ol>

<p>Now this terminology isn't fixed, and lots of people will talk about mate pairs when doing paired end, or vice versa. Caution is advised!  Also, I think Solid has some funky variations on this, but I haven't looked too closely.  And, the mate pair protocol is rather unreliable, IME.  Expect lots of non-mate-pair reads, and a wide range of insert sizes.</p>

<p>Oh, and I believe you can get mate pairs from 1.5K to 20K.  We just got Illumina PE reads at 500bp inserts, this was considered experimental by the company doing it.</p>

<ol>
<li><p>Removing duplicates can refer to several different things, but with all the second gen technologies, it is common to get a proportion of duplicate clones.  This can skew things, e.g. for de novo assembly, it will give an artificially high coverage of a region, and it might be incorrectly identified as a repeat.</p></li>
<li><p>The random placement of <em>sampe</em> is probably to make sure that you get the right coverage for repetitive regions. So it's orthogonal to removing duplicates that are sequencing artifacts. And if you have a dinucleotide region of (AT)<em>, a read of (AT)</em> and a read of (TA)* would not be considered duplicates, but be placed randomly.</p></li>
<li><p>Can't help you there, but Pierre seems to have it covered. :-)</p></li>
</ol>
