Daniel:Notebook/ComboLock/2016-9-27
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:
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 |