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.
File:Miseq20161110-ind30-pos1align-sam.png
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.
File:Miseq20161110-ind30-pos28align-sam.png
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.
Read Name | Ref Pos Start | CIGAR | Total Sequence | Offset | Seq1 | Seq2 | Seq3 | Seq4 | Seq5 | Seq6 | Seq7 |
Fragment Length-> | 121 | 29 | 8 | 18 | 8 | 18 | 10 | 18 | 6 | ||
Segment ID-> | AmpF | Barcode-Padlock0201 | Primer2RC | PCCB-RC | primer6RC | PolyT | PCCB-Template Match | Next 6 bp | |||
M00159:76:000000000-AV6P3:1:1108:13422:14156 | 28 | 55M66S | ACCACTCTCAGATGTTATCGAGGTCCGAC TCGCGCCACGCCGGTTACTGGGCG GGAGAGGTATCCCGATCACGCATCG CCGTTTTTTTTTCCGTTAGACACGGG ATCCGTGTACTCGTATG |
ACCACTCTCAGATGTTATCGAGGTCCGAC | TCGCGCCA | CGCCGGTTACTGGGCGGG | AGAGGTAT | CCCGATCACGCATCGCCG | TTTTTTTTTC | CGTTAGACACGGGATCCG | TGTACT |