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Search took 0.01 seconds. Search: Posts Made By: ajthomas
Forum: Sample Prep / Library Generation 06-01-2020, 09:41 AM
Replies: 5
Views: 3,705
Posted By ajthomas
There are a couple of options. The first is the...

There are a couple of options. The first is the SageHLS (suggested by ychang, above) https://sagescience.com/products/sagehls/.
The second is CAS9-targeted sequencing on the MinION. If you don't...
Forum: Illumina/Solexa 03-25-2020, 11:23 AM
Replies: 5
Views: 2,178
Posted By ajthomas
This is probably a bit late for you, but I'll...

This is probably a bit late for you, but I'll chime in here with my own experience for those who come after. I needed more indexes than were available in any Illumina product. However, rather than...
Forum: Illumina/Solexa 03-25-2020, 11:17 AM
Replies: 2
Views: 2,468
Posted By ajthomas
It sounds like you want to do something similar...

It sounds like you want to do something similar to 16S rRNA sequencing. That is a well-established protocol. I don't know what your amplicon is, but you can probably get some pretty good guidance...
Forum: Illumina/Solexa 03-25-2020, 09:49 AM
Replies: 1
Views: 1,465
Posted By ajthomas
I am a small sequencing core serving mostly just...

I am a small sequencing core serving mostly just my university, but I can run something for you. Send me a PM to discuss.
Forum: Bioinformatics 03-06-2018, 03:49 PM
Replies: 2
Views: 1,128
Posted By ajthomas
Maybe I'm missing something here, but it seems to...

Maybe I'm missing something here, but it seems to me that a de novo assembly followed by BLAST would be good for finding similar or matching contigs, but not so good at finding differences.

My...
Forum: Bioinformatics 03-02-2018, 10:10 AM
Replies: 2
Views: 1,128
Posted By ajthomas
find differences between two genomes

I have genomic sequencing data from two individuals. These are clones from the same cell line, but they have had a change made by homologous recombination. They should be identical, but one group of...
Forum: Ion Torrent 04-11-2016, 11:03 AM
Replies: 8
Views: 7,755
Posted By ajthomas
My preference for the Kapa kit is just a personal...

My preference for the Kapa kit is just a personal preference, not that I found it to work better. I didn't do any side-by-side comparison of the two or anything, I just used them both and found that...
Forum: Ion Torrent 03-01-2016, 11:22 AM
Replies: 8
Views: 7,755
Posted By ajthomas
The Ion Torrent-branded kits are much more...

The Ion Torrent-branded kits are much more expensive and I haven't found them to be any better than others. I've used the NEB kit and it works fine. More recently I tried the Kapa kit and I like it...
Forum: Ion Torrent 01-05-2016, 10:06 AM
Replies: 122
Views: 112,998
Posted By ajthomas
You don't absolutely need the Chef. We have the...

You don't absolutely need the Chef. We have the PGM and the Proton, but no Chef, and we get along just fine. The Chef automates some things and saves time, but it's not necessary. I guess what I'm...
Forum: Ion Torrent 09-30-2015, 02:55 PM
Replies: 7
Views: 7,634
Posted By ajthomas
Ion Torrent is the company/brand name. Ion...

Ion Torrent is the company/brand name. Ion Torrent's sequencers are the PGM and the Proton. Functionally they are very similar in that they use the same chemistry and principles. The primary...
Forum: Illumina/Solexa 09-25-2015, 02:10 PM
Replies: 145
Views: 322,276
Posted By ajthomas
I got those sequences from the copy of the letter...

I got those sequences from the copy of the letter that I downloaded about a month ago. It is dated August 2014 and is the current version available from Illumina today. If those are old sequences...
Forum: Illumina/Solexa 09-22-2015, 04:18 PM
Replies: 145
Views: 322,276
Posted By ajthomas
I've been trying to sort this out to order some...

I've been trying to sort this out to order some primers to use for making amplicon libraries on the Fluidigm Access Array. The Fluidigm protocol uses non-standard sequences (and therefore custom...
Forum: 454 Pyrosequencing 01-12-2015, 11:14 AM
Replies: 1
Views: 1,684
Posted By ajthomas
Are you thinking of combining them before making...

Are you thinking of combining them before making libraries, or after? I wouldn't combine them before making libraries for a couple of reasons. First, the cDNA will have to be double stranded if...
Forum: 454 Pyrosequencing 01-12-2015, 11:09 AM
Replies: 1
Views: 2,169
Posted By ajthomas
The problem with quantification using PicoGreen...

The problem with quantification using PicoGreen is that it will measure all the DNA is the sample, whether or not it can function in emPCR. Variation in ligation efficiency will therefore give you...
Forum: Ion Torrent 04-10-2014, 11:12 AM
Replies: 4
Views: 3,561
Posted By ajthomas
I've done something similar as well. In my case,...

I've done something similar as well. In my case, however, it was Lib-A library that I converted, so I used two different primers. I made one that used part of the Lib-A A adapter to switch it to...
Forum: The Pipeline 01-31-2014, 07:43 AM
Replies: 7
Views: 11,677
Posted By ajthomas
I looks like their technology might turn into...

I looks like their technology might turn into something useful at some point, but it's far from ready for prime time.
Forum: Bioinformatics 01-29-2014, 10:37 AM
Replies: 1
Views: 1,751
Posted By ajthomas
MIRA failure

I recently finished the first run on our new Ion Torrent Proton. The sample is genomic DNA from a non-model organism so I wanted to start with a de novo assembly using the Assembler plugin for...
Forum: RNA Sequencing 01-24-2014, 11:25 AM
Replies: 24
Views: 16,187
Posted By ajthomas
T7 (Eberwine) RNA amplification is commonly used...

T7 (Eberwine) RNA amplification is commonly used to amplify mRNA for analysis by microarrays, but I have not seen anyone use it to amplify RNA for RNA-Seq analysis. Is there any reason it wouldn't...
Forum: Ion Torrent 01-14-2014, 07:34 AM
Replies: 2
Views: 4,997
Posted By ajthomas
I doubt you would get any signal from ISPs left...

I doubt you would get any signal from ISPs left over from the previous run. They will have double-stranded DNA on them whereas the new ISPs will have single-stranded DNA with primers annealed. If...
Forum: 454 Pyrosequencing 12-02-2013, 11:36 AM
Replies: 4
Views: 2,974
Posted By ajthomas
No worries. I've been called worse.:)

No worries. I've been called worse.:)
Forum: 454 Pyrosequencing 12-02-2013, 08:44 AM
Replies: 4
Views: 2,974
Posted By ajthomas
I wouldn't sequence the beads if I were running...

I wouldn't sequence the beads if I were running it. The fact that you saw lots of short fragments indicates that even if there is sequence on those beads the sequence is no good. The information...
Forum: 454 Pyrosequencing 12-02-2013, 08:28 AM
Replies: 6
Views: 2,900
Posted By ajthomas
Some questions to help us understand so we can...

Some questions to help us understand so we can help you:

Are you talking about the .fna and .qual files that are automatically created as part of the process, or are these files you created later...
Forum: 454 Pyrosequencing 11-21-2013, 10:30 AM
Replies: 6
Views: 3,115
Posted By ajthomas
It's all of the reads that passed the filters. ...

It's all of the reads that passed the filters. It contains all of the same reads as the .sff file; the difference is that the .fna file contains only the trimmed sequence information rather than all...
Forum: 454 Pyrosequencing 11-08-2013, 08:23 AM
Replies: 3
Views: 2,307
Posted By ajthomas
I made some primers using the sequences found in...

I made some primers using the sequences found in the TCB (or is it an Application Note?) that describes unidirectional amplicon sequencing using the Lib-L primers. You can use the adapter sequences...
Forum: 454 Pyrosequencing 10-31-2013, 09:04 AM
Replies: 11
Views: 3,598
Posted By ajthomas
Are the key pass wells all control beads? If so,...

Are the key pass wells all control beads? If so, I would guess that you grabbed the wrong tube when you annealed the sequencing primer and used the enrichment primer instead.
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