Daniel:Notebook/ComboLock/2016-9-27

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Revision as of 20:58, 28 September 2016 by >Djacobse (→‎Methods-Blast)
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Positive Control X3 (Started 08/29/2016)

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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

File:Miseq20160912-BlastCountsHistogram.png