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Search: Posts Made By: AndrewP
Forum: Bioinformatics 04-03-2020, 08:40 AM
Replies: 244
Views: 105,819
Posted By AndrewP
I'm trying to use BBmap to find all perfect hits...

I'm trying to use BBmap to find all perfect hits or hits with an indel length 1.


bbmapskinner.sh in=kmer.fasta out=result.sam ambiguous=all strictmaxindel=1

I'm running a control experiment...
Forum: Sample Prep / Library Generation 04-05-2019, 05:17 PM
Replies: 3
Views: 787
Posted By AndrewP
Thanks for the advice, but I'm thinking more...

Thanks for the advice, but I'm thinking more along the lines of causing premature termination in the reverse transcriptase. I realize it's the opposite request of what most users need, but the...
Forum: Sample Prep / Library Generation 04-05-2019, 05:11 PM
Replies: 10
Views: 1,767
Posted By AndrewP
Maybe so, but it doesn't appear to be possible in...

Maybe so, but it doesn't appear to be possible in my hands using superscript II and the conditions from the SMART-seq2 protocol. Maybe some proprietary additives in the Clontech buffer makes a big...
Forum: Sample Prep / Library Generation 04-05-2019, 07:00 AM
Replies: 3
Views: 787
Posted By AndrewP
Need high number of short cDNA fragments

Hi all,

My application requires short cDNA fragments (up to 500 bp) with a defined 5' tag. I don't have the option to fragment RNA before reverse transcription using Mg and heat or the option to...
Forum: Sample Prep / Library Generation 04-05-2019, 06:00 AM
Replies: 10
Views: 1,767
Posted By AndrewP
In case anyone is trying something similar and...

In case anyone is trying something similar and finds this thread. Template switching is not possible in bacteria because they lack a 5' cap that RTase requires for terminal transferase activity. I...
Forum: Sample Prep / Library Generation 04-05-2019, 05:55 AM
Replies: 10
Views: 1,767
Posted By AndrewP
Most TS is at 42 degC which is the optimal...

Most TS is at 42 degC which is the optimal temperature for superscript II, I don't think the 5 degC makes much of a difference, but it fits in with other aspects of my workflow including better...
Forum: Sample Prep / Library Generation 03-04-2019, 05:35 AM
Replies: 6
Views: 4,562
Posted By AndrewP
I can't help much in that regard since I only...

I can't help much in that regard since I only work with microbial samples, but it should be clear from literature what most people use. I honestly don't think you need RNAlater, but I also don't...
Forum: Sample Prep / Library Generation 03-02-2019, 04:30 PM
Replies: 6
Views: 4,562
Posted By AndrewP
It should be fine considering it's just EDTA to...

It should be fine considering it's just EDTA to prevent RNA degradation and ammonium sulfate to precipitate proteins, and RNases in particular. You'll have to isolate RNA anyways, and that should get...
Forum: Sample Prep / Library Generation 03-01-2019, 10:20 AM
Replies: 10
Views: 1,767
Posted By AndrewP
Thanks for the advice, I'll give that a try. I...

Thanks for the advice, I'll give that a try. I should have done that the first time around, but was hesitant because of conditions of the experiment. Any thoughts on RT primer and TSO concentrations?...
Forum: Sample Prep / Library Generation 03-01-2019, 09:15 AM
Replies: 10
Views: 1,767
Posted By AndrewP
Thanks for the feedback cmbetts. I used the...

Thanks for the feedback cmbetts. I used the standard SSII buffer that has 3 mM MgCl2 because I've noticed some TS methods don't add additional MgCl2. I am thinking of adding 9 mM MgCl2 with 1M...
Forum: Sample Prep / Library Generation 03-01-2019, 06:58 AM
Replies: 10
Views: 1,767
Posted By AndrewP
Bacterial scRNA-seq experience?

Hi all,

I'm trying to perform a scRNA-like library prep in bacteria, so I cannot take advantage of the polyT reverse transcription primers. Instead, I'm using tagged random hexamers/decamers. I...
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