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====Alignment Positions==== Next I think it is important to get a picture of where the reads aligned. The following is a table created from the aligned reads by taking a tally of where on the reference (column 4) the reads aligned. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#92CDDC" width="150" height="30" | Alignment Position (on Reference) |style="background-color:#92CDDC" width="100" | Counts |style="background-color:#92CDDC" width="120" | Percent of Aligned Reads |style="background-color:#95B3D7" width="150" | Alignment Position (on Reference) |style="background-color:#95B3D7" width="100" | Counts |style="background-color:#95B3D7" width="120" | Percent of Aligned Reads |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 1 | align="center" | 386489 | align="center" | 80.4 |style="font-weight:bold" align="center" | 15 | align="center" | 1264 | align="center" | 0.3 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 2 | align="center" | 16587 | align="center" | 3.4 |style="font-weight:bold" align="center" | 16 | align="center" | 1192 | align="center" | 0.2 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 3 | align="center" | 11179 | align="center" | 2.3 |style="font-weight:bold" align="center" | 17 | align="center" | 1380 | align="center" | 0.3 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 4 | align="center" | 9185 | align="center" | 1.9 |style="font-weight:bold" align="center" | 18 | align="center" | 465 | align="center" | 0.1 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 5 | align="center" | 10926 | align="center" | 2.3 |style="font-weight:bold" align="center" | 19 | align="center" | 273 | align="center" | 0.1 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 6 | align="center" | 17120 | align="center" | 3.6 |style="font-weight:bold" align="center" | 20 | align="center" | 511 | align="center" | 0.1 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 7 | align="center" | 3906 | align="center" | 0.8 |style="font-weight:bold" align="center" | 21 | align="center" | 755 | align="center" | 0.2 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 8 | align="center" | 6500 | align="center" | 1.4 |style="font-weight:bold" align="center" | 22 | align="center" | 520 | align="center" | 0.1 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 9 | align="center" | 1034 | align="center" | 0.2 |style="font-weight:bold" align="center" | 23 | align="center" | 340 | align="center" | 0.0707 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 10 | align="center" | 4785 | align="center" | 1.0 |style="font-weight:bold" align="center" | 24 | align="center" | 179 | align="center" | 0.0372 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 11 | align="center" | 5689 | align="center" | 1.2 |style="font-weight:bold" align="center" | 25 | align="center" | 66 | align="center" | 0.0137 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 12 | align="center" | 911 | align="center" | 0.2 |style="font-weight:bold" align="center" | 26 | align="center" | 51 | align="center" | 0.0106 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 13 | align="center" | 2564 | align="center" | 0.5 |style="font-weight:bold" align="center" | 27 | align="center" | 33 | align="center" | 0.0069 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 14 | align="center" | 2805 | align="center" | 0.6 |style="font-weight:bold" align="center" | 28 | align="center" | 9 | align="center" | 0.0019 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 15 | align="center" | 1264 | align="center" | 0.3 |style="font-weight:bold" align="center" | 29 | align="center" | 1 | align="center" | 0.0002 |} From the table, it is clear that all of the reads align within the first 30bp of the reference, which corresponds to the AmpF region. The next step is to look at the alignment results more specifically. To do this I looked at the CIGAR strings (column 6) from the SAM table for different alignment positions. As an example I've included the first 10 alignments in the sam file. [[Image:Miseq20161110-ind30-pos1align-sam.png|1000px]] A few things stick out. First, all of the cigar strings show the match in the latter half of the reads. Second, all of the qualities here are actually poor in the first few bases rather than the last few. Next, we can look at the alignments near the far end. Since alignment 29 is only a single read (it is interesting, though) I've included the top results from alignment position 28 instead. [[Image:Miseq20161110-ind30-pos28align-sam.png|1000px]] Most of the reads have the same garbage present in the first base-aligned reads. However, look at the bottom read (there are several like it). This has the matching region in the first 50bp and has consistent high quality throughout the read. I will continue talking about these reads in the next section.
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