Brandon:LabNotes/Project1/2015-8-3
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Update of THS-seq protocol sequences to use Nextera XT V2 sequences
- THS-seq libraries generated with Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP) protocol have not been working on the MiSeq. Cluster density is really low, and when spiked in is 10-20X less then the total number of reads expected to be generated.
- PCR curves go up as expected, gels, gels size selection, RNA quants are all as expected. Tried replacing all reagents for RNA processing. Tried fresh GM12878 samples. Nothing got it to cluster on MiSeq.
- 2015-6-3 THS miseq troubleshooting R1
- 2015-6-10 THS miseq troubleshooting R2
- 2015-6-18 THS miseq troubleshooting R3
- 2015-7-1 THS miseq troubleshooting R4
- 2015-7-14 THS miseq troubleshooting R5
- However, these libraries worked on HiSeq, and 2015-04-01 did not work on miseq
- 2015-3-23 Guanidine HCl after transposition, taq2X and IVT on beads, 100 cell samples
- 2015-4-1 Tn5-059 versus ez-tn5 with THS-seq, using 500 cells and guanidine HCl method
- Called illumina and they said it is either sequencer not being cleaned right, or concentrations are off, or sequences in library generation protocol are not compatible. Considering currently old nextera sequences are being used from 5 years ago, and all current systems illumina sequencing systems do not use them, then maybe that is the problem.
- Thus will redesign THS-seq using Nextera XT V2 sequencing/index/flowcell/amplification primers.