Daniel:Notebook/ComboLock/2016-11-18: Difference between revisions

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[[Image:Miseq20161110-ind30-pos28align-sam.png|1000px]]
[[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.  
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.


===Individual Alignments====
===Individual Alignments====

Revision as of 18:55, 18 November 2016

Circularization Optimization Testing (Started 10-31-16)

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Sequencing Results

Sequenced Sample:Phusion-RCA product-Index 30 (Overnight Phusion) and Index 31 (2Hr Phusion Reaction)

Base Qualities

Prepared using Fastqc

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.

Individual Alignments=