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=Circularization Optimization Testing (Started [[Daniel:Notebook/ComboLock/2016-10-31|10-31-16]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Sequencing Results== '''Sequenced Sample:Phusion-RCA product-Index 30 (Overnight Phusion) and Index 31 (2Hr Phusion Reaction)''' ===Base Qualities=== Prepared using Fastqc <gallery perrow=2 heights=400px widths=400px title="Fastqc Base Qualities"> File:Miseq20161110-ind30-basequalities.png|Index 30 (Overnight Phusion) base qualities File:Miseq20161110-ind31-basequalities.png|Index 31 (2 Hour Phusion Rxn) base qualities </gallery> In both indexes the quality of the bases drops dramatically after about 55bp, although there are still some with high base qualities in the 3' end of the reads. These reads became important later as they seem to be the principle reads that I can make any sense of. ===Index 30 Sequence Analysis=== ====Overall Statistics==== From samtools idxstats (Total Reads-590207): AmplificationProducti-Forward 221 486719 0 Aligned reads: 82% ====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. ====Individual Alignments==== Now I'm going to look at the actual composition of these reads. There are two main ways I can do this. The first is manually by splitting up interesting reads in excel. This is painstaking manual labor but valuable for learning. The second is using blast. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#95B3D7;font-size:12pt;font-weight:bold" align="center" | width="200" height="31" | Read Name | width="85" | Ref Pos Start | width="100" | CIGAR | width="150" | Total Sequence | width="150" | Offset | width="80" | Seq1 | width="150" | Seq2 | width="85" | Seq3 | width="150" | Seq4 | width="85" | Seq5 | width="150" | Seq6 | width="65" | Seq7 |- style="font-size:12pt" | height="15" valign="bottom" | Fragment Length-> | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 121 | align="center" align="center" | 29 | align="center" align="center" | 8 | align="center" align="center" | 18 | align="center" align="center" | 8 | align="center" align="center" | 18 | align="center" align="center" | 10 | align="center" align="center" | 18 | align="center" align="center" | 6 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Segment ID-> | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | | align="center" | AmpF | align="center" | Barcode-Padlock0201 | align="center" | Primer2RC | align="center" | PCCB-RC | align="center" | primer6RC | align="center" | PolyT | align="center" | PCCB-Template Match | align="center" | Next 6 bp |- style="font-size:12pt" | height="100" valign="bottom" | M00159:76:000000000-AV6P3:1:1108:13422:14156 | align="center" align="center" | 28 | align="center" | 55M66S | align="center" | ACCACTCTCAGATGTTATCGAGGTCCGAC<br>TCGCGCCACGCCGGTTACTGGGCG<br>GGAGAGGTATCCCGATCACGCATCG<br>CCGTTTTTTTTTCCGTTAGACACGGG<br>ATCCGTGTACTCGTATG | align="center" | ACCACTCTCAGATGTTATC<br>GAGGTCCGAC | align="center" | TCGCGCCA | align="center" | CGCCGGTTACTGGGCGGG | align="center" | AGAGGTAT | align="center" | CCCGATCACGCATCGCCG | align="center" | TTTTTTTTTC | align="center" | CGTTAGACACGGGATCCG | align="center" | TGTACT |} The most important part here is that the bridge seems to be forming between the PCCB probe and the padlock. The circular product does not appear to contain the latch or A probe at all, but only contains the B probe and padlock. ===Index 31 Sequence Analysis=== ====Overall Statistics==== From samtools idxstats (Total Reads-1349673): AmplificationProducti-Forward 221 1053240 0 Alignment Percentage: 78% ====Alignment Positions==== {| 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" | 828175 | align="center" | 78.6 |style="font-weight:bold" align="center" | 15 | align="center" | 2638 | 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" | 36858 | align="center" | 3.5 |style="font-weight:bold" align="center" | 16 | align="center" | 2640 | align="center" | 0.3 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 3 | align="center" | 24997 | align="center" | 2.4 |style="font-weight:bold" align="center" | 17 | align="center" | 2867 | 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" | 21234 | align="center" | 2.0 |style="font-weight:bold" align="center" | 18 | align="center" | 984 | align="center" | 0.1 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 5 | align="center" | 24639 | align="center" | 2.3 |style="font-weight:bold" align="center" | 19 | align="center" | 540 | 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" | 37771 | align="center" | 3.6 |style="font-weight:bold" align="center" | 20 | align="center" | 1147 | align="center" | 0.1 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 7 | align="center" | 9018 | align="center" | 0.9 |style="font-weight:bold" align="center" | 21 | align="center" | 1641 | 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" | 14488 | align="center" | 1.4 |style="font-weight:bold" align="center" | 22 | align="center" | 1273 | align="center" | 0.1 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 9 | align="center" | 2144 | align="center" | 0.2 |style="font-weight:bold" align="center" | 23 | align="center" | 851 | align="center" | 0.1 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 10 | align="center" | 11316 | align="center" | 1.1 |style="font-weight:bold" align="center" | 24 | align="center" | 469 | align="center" | 0.045 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 11 | align="center" | 12873 | align="center" | 1.2 |style="font-weight:bold" align="center" | 25 | align="center" | 151 | align="center" | 0.014 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 12 | align="center" | 2013 | align="center" | 0.2 |style="font-weight:bold" align="center" | 26 | align="center" | 130 | align="center" | 0.012 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 13 | align="center" | 6004 | align="center" | 0.6 |style="font-weight:bold" align="center" | 27 | align="center" | 83 | align="center" | 0.008 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 14 | align="center" | 6274 | align="center" | 0.6 |style="font-weight:bold" align="center" | 28 | align="center" | 16 | align="center" | 0.002 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" align="center" height="15" | 15 | align="center" | 2638 | align="center" | 0.3 |style="font-weight:bold" align="center" | 29 | align="center" | 6 | align="center" | 0.001 |} Once again, the reads all align to the first 30bp (to start). So let's look at them more closely. The following are the reads aligned to position 1 of the fasta reference (completed amplicon from PCR). [[Image:Miseq20161110-ind31-pos1align-sam.png|1000px]] Now let's look at the bp28 aligned reads again. [[Image:Miseq20161110-ind31-pos28align-sam-highlight.png|1000px]] Aaaaaand we can see that again, many of the reads contain only the AmpF signal, but a few of the reads (designated by the CIGAR string with matching bases at the beginning, highlighted) are interesting. In the next section I will cover the composition of those sequences. ====Individual Alignments==== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#95B3D7;font-size:12pt;font-weight:bold" align="center" | width="200" height="31" | Read Name | width="85" | Ref Pos Start | width="100" | CIGAR | width="150" | Total Sequence | width="150" | Offset | width="80" | Seq1 | width="150" | Seq2 | width="85" | Seq3 | width="150" | Seq4 | width="85" | Seq5 | width="150" | Seq6 | width="85" | Seq7 |- style="font-size:12pt" | height="15" valign="bottom" | Fragment Length-> | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 121 | align="center" align="center" | 29 | align="center" align="center" | 8 | align="center" align="center" | 18 | align="center" align="center" | 8 | align="center" align="center" | 18 | align="center" align="center" | 10 | align="center" align="center" | 18 | align="center" align="center" | 6 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Segment ID-> | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | | align="center" | AmpF | align="center" | Barcode-Padlock0201 | align="center" | Primer2RC | align="center" | PCCB-RC | align="center" | primer6RC | align="center" | PolyT | align="center" | PCCB-Template Match | align="center" | Next 6 bp |- style="font-size:12pt" | height="45" valign="bottom" | M00159:76:000000000-AV6P3:1:2115:23149:21858 | align="center" align="center" | 28 | align="center" | 55M66S | valign="bottom" | ACCACTCTCAGATGTT ATCGAGGTCCGACTCGC GCCACGCCGGTTACTGGGC GGGAGAGGTATCCCGATC ACGCATCGCCGTTTT TTTTTTCGTTAGA CACGGGATCCGTGTAT AAGTTGA | align="center" | ACCACTCTCAGATGTTAT CGAGGTCCGAC | align="center" | TCGCGCCA | align="center" | CGCCGGTTACTGGGCGGG | align="center" | AGAGGTAT | align="center" | CCCGATCACGCATCGCCG | align="center" | TTTTTTTTTT | align="center" | CGTTAGACACGGGATCCG | align="center" | TGTATA |} {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#95B3D7;font-size:12pt;font-weight:bold" align="center" | width="200" height="31" | Read Name | width="85" | Ref Pos Start | width="85" | CIGAR | width="150" | Total Sequence | width="150" | Offset | width="85" | Seq1 | width="150" | Seq2 | width="85" | Seq3 | width="150" | Seq4 | width="85" | Seq5 | width="150" | Seq6 | width="85" | Seq7 |- style="font-size:12pt" | height="15" valign="bottom" | Fragment Length-> | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | 121 | align="center" align="center" | 29 | align="center" align="center" | 8 | align="center" align="center" | 18 | align="center" align="center" | 8 | align="center" align="center" | 18 | align="center" align="center" | 10 | align="center" align="center" | 18 | align="center" align="center" | 6 |- style="background-color:#BFBFBF;font-size:12pt" | height="30" valign="bottom" | Segment ID-> | align="center" align="center" | | align="center" align="center" | | align="center" align="center" | | align="center" | AmpF | align="center" | Barcode-Padlock0201 | align="center" | Primer2RC | align="center" | PCCB-RC | align="center" | primer6RC | align="center" | PolyT | align="center" | PCCB-Template Match | align="center" | Next 6 bp |- style="font-size:12pt" | height="45" valign="bottom" | M00159:76:000000000-AV6P3:1:1115:16510:5351 | align="center" align="center" | 28 | align="center" | 55M66S | valign="bottom" | ACCACTCTCAGATGTTATCGA GGTCCGACTCGCGCCACGCCGGT TACTGGGCGGGAGAGG TATCCCGATCACGCATCGC CGTTTTTTTTTTCGTTAGA CACGGGATCCGTGTATCTATCCC | align="center" | ACCACTCTCAGATGTTATCG AGGTCCGAC | align="center" | TCGCGCCA | align="center" | CGCCGGTTACTGGGCGGG | align="center" | AGAGGTAT | align="center" | CCCGATCACGCATCGCCG | align="center" | TTTTTTTTTT | align="center" | CGTTAGACACGGGATCCG | align="center" | TGTATC |} ==Concluding Remarks== While there are definitely circular products going on here, there is no question that the circular product appears to have the wrong makeup. Just in case, I am sequencing the reads again in a new run, along with a cells result that I am also sequencing. Based on the results from [[Daniel:Notebook/ComboLock/2016-11-16|Wednesday]] I wouldn't be surprised if the cell results are also not great. [[Category:ComboLock]] [[Category:20161031]]
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