Daniel:Notebook/ComboLock/2016-9-27: Difference between revisions

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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| 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="226" height="28" | Name
| width="65" | Read 1/2
| width="283" | Sequence
| width="65" | Offset
| width="114" | Seq 1
| width="65" | Seq1 Match
| width="82" | Index34? (R1)
| width="108" | Seq2
| width="146" | Seq 2 Match
| width="165" | Seq3
|- style="font-size:12pt"  valign="bottom"
|- style="font-size:12pt"  valign="bottom"
| width="183" height="45" | @M00159:51:000000000-ATU5U:1:1101:15771:2370 1:N:0:AACTGA
| height="30" | @M00159:51:000000000-ATU5U:1:1101:15771:2370 1:N:0:AACTGA
| width="55" | R1
| R1
| width="276" | GTGTTTGCGGCTAAGCGTTCCTAGCNANNGAATCTCGTATGCCGTCTTCTGCNNGCACAAAAAAACTCTTTTTNC
| GTGTTTGCGGCTAAGCGTTCCTAGCNANNGAATCTCGTATGCCGTCTTCTGCNNGCACAAAAAAACTCTTTTTNC
| width="110" | GT
| GT
| width="103" | GTTTGCGGCTAAGCGTTCCTAGC
| GTTTGCGGCTAAGCGTTCCTAGC
| width="90" | AmpR6.3-RevComp
| AmpR6.3-RevComp
| width="65" | NANNGA
| NANNGA
| width="196" | ATCTCGTATGCCGTCTTCTGCNNGC
| ATCTCGTATGCCGTCTTCTGCNNGC
| width="120" | AmpR6.3-RevComp bp: 33-53
| AmpR6.3-RevComp bp: 33-53
| width="120" | ACAAAAAAACTCTTT
| ACAAAAAAACTCTTTTTNC
| width="65" | TTNC


|- style="font-size:12pt"  valign="bottom"
|- style="background-color:#D9D9D9;font-size:12pt"  valign="bottom"
| height="45" | @M00159:51:000000000-ATU5U:1:1101:15771:2370 2:N:0:AACTGA
| height="30" | @M00159:51:000000000-ATU5U:1:1101:15771:2370 2:N:0:AACTGA
  | R2
  | R2
  | ACTTCGGACCTCGATAACATTTGAGAGTGGTGTAGATCTTGATGTTCGCCGAATCNTTAAAAANAAATGTNTTCNT
  | ACTTCGGACCTCGATAACATTTGAGAGTGGTGTAGATCTTGATGTTCGCCGAATCNTTAAAAANAAATGTNTTCNT
Line 65: Line 76:
  | GTAGATCTTGATGTTCGCCGAATCN
  | GTAGATCTTGATGTTCGCCGAATCN
  | First 11 match AmpF6.4Rev Comp 30-40
  | First 11 match AmpF6.4Rev Comp 30-40
  | TTAAAAANAAATGTN
  | TTAAAAANAAATGTNTTCN
| TTCNT


|}
|}

Revision as of 20:38, 28 September 2016

Positive Control X3 (Started 08/29/2016)

Back to Calendar

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

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:

Name Read 1/2 Sequence Offset Seq 1 Seq1 Match Index34? (R1) Seq2 Seq 2 Match Seq3
@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 ACAAAAAAACTCTTTTTNC
@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 TTAAAAANAAATGTNTTCN

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