update to Kit12 chemistry?

[tamara-nano]

Hey Simone,

I was wondering if you're planning to update your wonderful pipeline to the new kit12/14 chemistry, flowcells and super accuracy basecalling? :-)

With best regards, Tamara

[MaestSi]

Ciao Tamara, I think I may only have to introduce some adjustments in the preprocessing step (MinION_mobile_lab.R), while the remaining scripts should work as they are, just adjusting parameters to make the alignment more stringent. Actually I never tested the pipeline on such data, but if you are able to provide me with a raw fast5 file with a couple thousand reads, I'm happy to test it. Best, Simone

[tamara-nano]

Yeah, i've got some test-files with Zymobiomics DNA Microbiome Standard from kit12 with standard 27F and 1492R primer (not barcoded). The whole run has 40gb of which each fast5 file has ~100mb. Let me know how much from it you would need and how to provide them to you. Best, Tamara

[MaestSi]

I think one single fast5 file should be enough to test! I think Google Drive (or whatever you prefer) should work. Best, Simone

[MaestSi]

Ciao Tamara, I just slightly updated the preprocessing pipeline to enable demultiplexing to be skipped and adjusted some parameters. With the relevant parameters reported below, I tested the pipeline on the fast5 you sent me.

kit <- "SQK-LSK112"
flowcell <- "FLO-MIN112"
conf_basecalling_flag <- 1
conf_par_basecalling <- "-c dna_r10.4_e8.1_hac.cfg -x 'auto' --gpu_runners_per_device 1"
skip_demultiplexing_flag <- 1
min_seq_length <- 1200
max_seq_length <- 1600
primers_length <- 25
CLASSIFIER <- "Vsearch"
MAX_ACCEPTS <- 3
QUERY_COV <- 0.8
ID_THR <- 0.90

Atttached, you can find the taxa barplot I obtained. You can unzip it and upload it to QIIME2 View: taxa-bar-plots.qzv.zip Of course you can play around a little bit with parameters. In case, please let me know about your tests! Best, Simone