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Forum: Bioinformatics 05-06-2020, 10:01 AM
Replies: 0
Views: 177
Posted By seqtechno1
Smile Windows Ubuntu, Kallisto, R, sleuth, conda, bioconda, miniconda, bioconductor

Hello everyone,

I'm trying to sort out getting sleuth working with R. I currently have windows ubuntu subshell running and have installed conda, bioconda, bioconductor, R, kallisto, and sleuth....
Forum: RNA Sequencing 05-16-2019, 07:42 AM
Replies: 1
Views: 2,263
Posted By seqtechno1
Question high percentage of reads mapping to tRNA?

How do you interpret this? What does it mean for wet-lab side of things, does it inform any step that went right or wrong? This is for a total RNAseq minus rRNA experiment....
Forum: Bioinformatics 12-07-2018, 05:34 AM
Replies: 1
Views: 1,146
Posted By seqtechno1
Smile demultiplexing multiple runs with and without samples same sample sheet

Hello all,

I'm going to demultiplex a run with a sample sheet in which there are entries and adapter sequences in the sheet with no corresponding physical samples in the pool. I'm doing this...
Forum: Sample Prep / Library Generation 10-26-2018, 05:46 AM
Replies: 1
Views: 1,354
Posted By seqtechno1
Smile Comprehensive uv/vis spectrophotometry absorption library?

Hi all,

is anyone aware of a database where one can go and search for a compound by name? I have contaminants I cannot remove from RNAs after column purification, and I want to check known...
Forum: Sample Prep / Library Generation 07-11-2018, 07:04 AM
Replies: 2
Views: 1,662
Posted By seqtechno1
hi austinso, thanks for the information....

hi austinso,

thanks for the information. i'm going to run through the calculations so that i understand the math better. i will have enough for sequencing because i can normalize all of my...
Forum: Sample Prep / Library Generation 07-10-2018, 08:12 AM
Replies: 2
Views: 1,662
Posted By seqtechno1
Smile ddPCR for quantification of Illumina TruSeq library

Hello,

I am planning on using the BioRad Truseq ddPCR assay for Illumina quantification of a truseq cDNA library I made from RNA. The protocol calls for running dilutions of library in 10^-6,...
Forum: Core Facilities 06-06-2018, 07:25 AM
Replies: 3
Views: 4,871
Posted By seqtechno1
pacbio sequel system

how much do core facilities charge to run the instrument, not per sample or SMRTcell but just to recover the cost of running the device? (electricity, nitrogen, etc.)
Forum: Core Facilities 06-06-2018, 07:11 AM
Replies: 1
Views: 3,839
Posted By seqtechno1
yes

we have the same exact problem. its just meant to make more money for agilent sadly. have yet to find a workaround
Forum: Sample Prep / Library Generation 05-30-2018, 06:06 AM
Replies: 1
Views: 1,367
Posted By seqtechno1
Thermal cycler with variable temperature lid?

Does this machine exist:, for example, a machine that can change the lid temperature during the course of the reactions? Something tells me that having the lid set to 75C when my reverse...
Forum: Sample Prep / Library Generation 05-10-2018, 12:19 PM
Replies: 6
Views: 2,690
Posted By seqtechno1
Smile Is Qubit good for multiple samples?

Hello,

We've been having some quantitation issues recently, I'm wondering whether the Qubit fluorometers are good for processing lots of samples (96 at a time?)
Forum: RNA Sequencing 11-08-2017, 06:36 AM
Replies: 5
Views: 2,206
Posted By seqtechno1
Hi HESmith. thanks for your input. the library...

Hi HESmith. thanks for your input. the library construction finishes with two separate ampure xp bead purifications. do you think it is worth performing again?
Forum: RNA Sequencing 11-08-2017, 06:23 AM
Replies: 5
Views: 2,206
Posted By seqtechno1
Smile strong 140bp peak following RNA library construction

Hello all,
I have created an illumina truseq library on bacteria by first performing ribozero rRNA depletion kit and then performing a truseq stranded mRNA prep while skipping the poly-A selection...
Forum: Introductions 10-16-2017, 06:14 AM
Replies: 3
Views: 1,065
Posted By seqtechno1
concentrating DNA

Do a concentration and purification using AMPure XP magnetic beads (they are SPRI beads, you can google how they work if interested) and concentrate and purify the DNA using these and a magnetic...
Forum: Sample Prep / Library Generation 10-11-2017, 04:57 AM
Replies: 4
Views: 3,227
Posted By seqtechno1
--------------------------------------------------...

--------------------------------------------------------------------

so how would you perform RNAseq on bacterial samples from human? Poly-A selection is not always an option....
Forum: RNA Sequencing 10-09-2017, 12:26 PM
Replies: 2
Views: 1,396
Posted By seqtechno1
Angry Single kit for bacterial RNAseq projects (from clinical samples)?

Hello,

Does anyone know of a standalone kit or protocol which could deplete mammalian rRNA and bacterial rRNA to be left with bacterial RNA we want for sequencing? The best way I know how...
Forum: RNA Sequencing 10-09-2017, 08:41 AM
Replies: 2
Views: 1,368
Posted By seqtechno1
Forum: Sample Prep / Library Generation 08-24-2017, 10:49 AM
Replies: 2
Views: 2,778
Posted By seqtechno1
troubleshooting agilent bioanalyzer error

I ran an RNA 6000 nano chip with RNA samples in all 12 wells, some randomly came back flagged with error: unexpected signal in fast region. Is there any way to troubleshoot this after the fact to get...
Forum: Sample Prep / Library Generation 08-14-2017, 08:12 AM
Replies: 7
Views: 3,001
Posted By seqtechno1
electropherogram error

Hello,
trying to be of some assistance here, what were your errors? If you look on the simulated gel to the right there is a red dot, which indicates an error has occurred, whether some peak on...
Forum: Sample Prep / Library Generation 08-09-2017, 04:59 AM
Replies: 0
Views: 934
Posted By seqtechno1
Low input of primers for pre optimized PCR step

Hello,
I have created a situation where I only have enough reagents to add approximately 1/5 of the primers recommended (pre optimized by Illumina) for a library amplification step (to each well...
Forum: Pacific Biosciences 05-26-2017, 06:30 AM
Replies: 2
Views: 2,640
Posted By seqtechno1
bluepippin size selection for SMRTbells

Hello,

at which step of the 10kb protocol for preparation of SMRTbell libraries do you run BluePippin 20kb cassette for size selection? I am preparing gDNAs for complete sequencing.
Forum: Pacific Biosciences 05-22-2017, 10:45 AM
Replies: 2
Views: 2,572
Posted By seqtechno1
Unhappy PacBio reagents

If a box of PacBio polymerase binding reagents is left at room temperature and allowed to warm to ambient temperature is it now useless?
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