Daniel:Notebook/ComboLock/2016-11-18
Circularization Optimization Testing (Started 10-31-16)
Sequencing Results
Sequenced Sample:Phusion-RCA product-Index 30 (Overnight Phusion) and Index 31 (2Hr Phusion Reaction)
Base Qualities
Prepared using Fastqc
- Miseq20161110-ind30-basequalities.png
Index 30 (Overnight Phusion) base qualities
- Miseq20161110-ind31-basequalities.png
Index 31 (2 Hour Phusion Rxn) base qualities
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.
Alignment Position (on Reference) | Counts | Percent of Aligned Reads | Alignment Position (on Reference) | Counts | Percent of Aligned Reads |
1 | 386489 | 80.4 | 15 | 1264 | 0.3 |
2 | 16587 | 3.4 | 16 | 1192 | 0.2 |
3 | 11179 | 2.3 | 17 | 1380 | 0.3 |
4 | 9185 | 1.9 | 18 | 465 | 0.1 |
5 | 10926 | 2.3 | 19 | 273 | 0.1 |
6 | 17120 | 3.6 | 20 | 511 | 0.1 |
7 | 3906 | 0.8 | 21 | 755 | 0.2 |
8 | 6500 | 1.4 | 22 | 520 | 0.1 |
9 | 1034 | 0.2 | 23 | 340 | 0.0707 |
10 | 4785 | 1.0 | 24 | 179 | 0.0372 |
11 | 5689 | 1.2 | 25 | 66 | 0.0137 |
12 | 911 | 0.2 | 26 | 51 | 0.0106 |
13 | 2564 | 0.5 | 27 | 33 | 0.0069 |
14 | 2805 | 0.6 | 28 | 9 | 0.0019 |
15 | 1264 | 0.3 | 29 | 1 | 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.