Daniel:Notebook/ComboLock/2016-9-27: Difference between revisions
>Djacobse (Created page with "=Positive Control X3 (Started 08/29/2016)= Back to Calendar Sequencing data in Genome miner at /media/N...") |
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File:2016-09-05-X3Dilution-SizeSelect-After.png|After Image | File:2016-09-05-X3Dilution-SizeSelect-After.png|After Image | ||
</gallery> | </gallery> | ||
===Methods-Manual Inspection=== | |||
I used several methods to analyze this data set. For the simplest, I just took the raw sequences and put them into excel charts. Example for one read below: | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" valign="bottom" | |||
| width="183" height="45" | @M00159:51:000000000-ATU5U:1:1101:15771:2370 1:N:0:AACTGA | |||
| width="55" | R1 | |||
| width="276" | GTGTTTGCGGCTAAGCGTTCCTAGCNANNGAATCTCGTATGCCGTCTTCTGCNNGCACAAAAAAACTCTTTTTNC | |||
| width="110" | GT | |||
| width="103" | GTTTGCGGCTAAGCGTTCCTAGC | |||
| width="90" | AmpR6.3-RevComp | |||
| width="65" | NANNGA | |||
| width="196" | ATCTCGTATGCCGTCTTCTGCNNGC | |||
| width="120" | AmpR6.3-RevComp bp: 33-53 | |||
| width="120" | ACAAAAAAACTCTTT | |||
| width="65" | TTNC | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="45" | @M00159:51:000000000-ATU5U:1:1101:15771:2370 2:N:0:AACTGA | |||
| R2 | |||
| ACTTCGGACCTCGATAACATTTGAGAGTGGTGTAGATCTTGATGTTCGCCGAATCNTTAAAAANAAATGTNTTCNT | |||
| AC | |||
| TTCGGACCTCGATAACATTTGAG | |||
| AmpF6.4-RevComp | |||
| AGTGGT | |||
| GTAGATCTTGATGTTCGCCGAATCN | |||
| First 11 match AmpF6.4Rev Comp 30-40 | |||
| TTAAAAANAAATGTN | |||
| TTCNT | |||
|} | |||
These allow me to manually assess the sequences, but are also not great for larger scale processing (I only did ~4 for any given index). For larger scale analyses, I used blast. | |||
===Methods-Blast=== | |||
To do blast, I used a database made of the various primer, adapter, and index sequences. I set a threshold of 80% match to get rid of small alignments (that is, the read must contain >=80% of the bases matching to the primer/adapter/index). The table below summarizes the sequences targeted and how many bp a match requires. | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B7DEE8;font-size:12pt;font-weight:bold" align="center" | |||
| width="86" height="45" | Name | |||
| width="65" | Length | |||
| width="101" | Number of bp Correct for 80% match | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | primer2 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | primer4 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | primer6 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | primer12 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | IlluminaP5 | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 16 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | IlluminaP7 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 20 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | LinkerL | |||
| align="center" align="center" valign="bottom" | 23 | |||
| align="center" align="center" valign="bottom" | 19 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | LinkerR | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 20 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Latch1Barcode | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Latch2Barcode | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Latch3Barcode | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Latch6Barcode | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Latch8Barcode | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Latch9Barcode | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|} | |||
[[Category:ComboLock]] [[Category:20160829]] |
Revision as of 19:16, 28 September 2016
Positive Control X3 (Started 08/29/2016)
Sequencing data in Genome miner at /media/NAS3_volume2/Dan/combolock/miseq_20160912
Data Analysis
As a reminder, here is a table of the three samples, as well as the size select gels for the library prep.
Sample | Index | Approx bp Size |
Sample0 | 34 | 130 |
Sample1 | 35 | >500 |
Sample2 | 36 | 100 |
- 2016-09-05-X3Dilution-SizeSelect.png
Before Image
- 2016-09-05-X3Dilution-SizeSelect-After.png
After Image
Methods-Manual Inspection
I used several methods to analyze this data set. For the simplest, I just took the raw sequences and put them into excel charts. Example for one read below:
@M00159:51:000000000-ATU5U:1:1101:15771:2370 1:N:0:AACTGA | R1 | GTGTTTGCGGCTAAGCGTTCCTAGCNANNGAATCTCGTATGCCGTCTTCTGCNNGCACAAAAAAACTCTTTTTNC | GT | GTTTGCGGCTAAGCGTTCCTAGC | AmpR6.3-RevComp | NANNGA | ATCTCGTATGCCGTCTTCTGCNNGC | AmpR6.3-RevComp bp: 33-53 | ACAAAAAAACTCTTT | TTNC |
@M00159:51:000000000-ATU5U:1:1101:15771:2370 2:N:0:AACTGA | R2 | ACTTCGGACCTCGATAACATTTGAGAGTGGTGTAGATCTTGATGTTCGCCGAATCNTTAAAAANAAATGTNTTCNT | AC | TTCGGACCTCGATAACATTTGAG | AmpF6.4-RevComp | AGTGGT | GTAGATCTTGATGTTCGCCGAATCN | First 11 match AmpF6.4Rev Comp 30-40 | TTAAAAANAAATGTN | TTCNT |
These allow me to manually assess the sequences, but are also not great for larger scale processing (I only did ~4 for any given index). For larger scale analyses, I used blast.
Methods-Blast
To do blast, I used a database made of the various primer, adapter, and index sequences. I set a threshold of 80% match to get rid of small alignments (that is, the read must contain >=80% of the bases matching to the primer/adapter/index). The table below summarizes the sequences targeted and how many bp a match requires.
Name | Length | Number of bp Correct for 80% match |
primer2 | 18 | 15 |
primer4 | 18 | 15 |
primer6 | 18 | 15 |
primer12 | 18 | 15 |
IlluminaP5 | 20 | 16 |
IlluminaP7 | 24 | 20 |
LinkerL | 23 | 19 |
LinkerR | 25 | 20 |
Latch1Barcode | 8 | 7 |
Latch2Barcode | 8 | 7 |
Latch3Barcode | 8 | 7 |
Latch6Barcode | 8 | 7 |
Latch8Barcode | 8 | 7 |
Latch9Barcode | 8 | 7 |