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

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Circularization Optimization Testing (Started 10-31-16)= Back to Calendar ==Sequencing Results== '''...")
 
>Djacobse
Line 15: Line 15:
File:Miseq20161110-ind31-basequalities.png|Index 31 (2 Hour Phusion Rxn) base qualities
File:Miseq20161110-ind31-basequalities.png|Index 31 (2 Hour Phusion Rxn) base qualities
</gallery>
</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="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center"
| width="103" height="30" | Alignment Position (on Reference)
| width="91" | Counts
| width="99" | Percent of Aligned Reads
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 1
| align="center" | 386489
| align="center" | 79.4
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 2
| align="center" | 16587
| align="center" | 3.4
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 3
| align="center" | 11179
| align="center" | 2.3
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 4
| align="center" | 9185
| align="center" | 1.9
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 5
| align="center" | 10926
| align="center" | 2.2
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 6
| align="center" | 17120
| align="center" | 3.5
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 7
| align="center" | 3906
| align="center" | 0.8
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 8
| align="center" | 6500
| align="center" | 1.3
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 9
| align="center" | 1034
| align="center" | 0.2
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 10
| align="center" | 4785
| align="center" | 1.0
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 11
| align="center" | 5689
| align="center" | 1.2
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 12
| align="center" | 911
| align="center" | 0.2
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 13
| align="center" | 2564
| align="center" | 0.5
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 14
| align="center" | 2805
| align="center" | 0.6
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 15
| align="center" | 1264
| align="center" | 0.3
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 16
| align="center" | 1192
| align="center" | 0.2
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 17
| align="center" | 1380
| align="center" | 0.3
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 18
| align="center" | 465
| align="center" | 0.1
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 19
| align="center" | 273
| align="center" | 0.1
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 20
| align="center" | 511
| align="center" | 0.1
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 21
| align="center" | 755
| align="center" | 0.2
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 22
| align="center" | 520
| align="center" | 0.1
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 23
| align="center" | 340
| align="center" | 0.0699
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 24
| align="center" | 179
| align="center" | 0.0368
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 25
| align="center" | 66
| align="center" | 0.0136
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 26
| align="center" | 51
| align="center" | 0.0105
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 27
| align="center" | 33
| align="center" | 0.0068
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 28
| align="center" | 9
| align="center" | 0.0018
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | 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|800px]]




[[Category:ComboLock]] [[Category:20161031]]
[[Category:ComboLock]] [[Category:20161031]]

Revision as of 18:41, 18 November 2016

Circularization Optimization Testing (Started 10-31-16)

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

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
1 386489 79.4
2 16587 3.4
3 11179 2.3
4 9185 1.9
5 10926 2.2
6 17120 3.5
7 3906 0.8
8 6500 1.3
9 1034 0.2
10 4785 1.0
11 5689 1.2
12 911 0.2
13 2564 0.5
14 2805 0.6
15 1264 0.3
16 1192 0.2
17 1380 0.3
18 465 0.1
19 273 0.1
20 511 0.1
21 755 0.2
22 520 0.1
23 340 0.0699
24 179 0.0368
25 66 0.0136
26 51 0.0105
27 33 0.0068
28 9 0.0018
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