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Forum: Sample Prep / Library Generation 03-27-2020, 10:23 AM
Replies: 12
Views: 2,978
Posted By MU Core
That's correct, a single bead clean-up on the...

That's correct, a single bead clean-up on the pool. As long as you have good amplification across the plate and normalize template input amount, there is no need to quantitate and normalize the PCR...
Forum: Sample Prep / Library Generation 03-26-2020, 05:53 AM
Replies: 12
Views: 2,978
Posted By MU Core
Do you normalize your template input for the 1st...

Do you normalize your template input for the 1st PCR? We have found this very useful and critical in getting an even representation of reads in the pool. As long as there are no PCR inhibitors in...
Forum: Sample Prep / Library Generation 11-12-2019, 05:45 AM
Replies: 21
Views: 7,813
Posted By MU Core
For those seeking a bacterial rRNA depletion kit,...

For those seeking a bacterial rRNA depletion kit, NEB has just released a kit that works very well in our hands.
...
Forum: Sample Prep / Library Generation 02-01-2019, 04:50 AM
Replies: 21
Views: 7,813
Posted By MU Core
Yes, final reads which are rRNA.

Yes, final reads which are rRNA.
Forum: Sample Prep / Library Generation 01-31-2019, 07:21 AM
Replies: 21
Views: 7,813
Posted By MU Core
Our group has obtained similar results with the...

Our group has obtained similar results with the RiboMinus kit. rRNA mapping at ~80%. Alternatives are still needed.
Forum: Illumina/Solexa 10-08-2018, 04:49 AM
Replies: 3
Views: 1,392
Posted By MU Core
I would be happy to talk with you in detail about...

I would be happy to talk with you in detail about your sequencing needs off-line. Contact info can be found at the website below.

https://dnacore.missouri.edu/
Forum: Sample Prep / Library Generation 05-04-2018, 04:47 AM
Replies: 13
Views: 4,565
Posted By MU Core
chanwu, We trust both measurements but use...

chanwu,

We trust both measurements but use each for different library types. qPCR is used for PCR-free library preps with all other libraries which undergo amplification being measured by Qubit. ...
Forum: Illumina/Solexa 11-01-2017, 04:54 AM
Replies: 13
Views: 1,979
Posted By MU Core
Is there a possibility of adapter dimers? May...

Is there a possibility of adapter dimers? May need to perform an additional library clean-up.
Forum: Illumina/Solexa 08-29-2017, 05:11 AM
Replies: 22
Views: 7,301
Posted By MU Core
Joanna, looks to me as though you've resolved the...

Joanna, looks to me as though you've resolved the library issue with the new primers. I would agree that this particular run is over-clustered. We observe similar quality issues when 16S libraries...
Forum: Illumina/Solexa 05-25-2017, 05:18 AM
Replies: 23
Views: 9,473
Posted By MU Core
I've been told that Illumina will soon (~1-2...

I've been told that Illumina will soon (~1-2 months) be releasing a kit with 96 dual-unique indexes.
Forum: RNA Sequencing 05-17-2017, 04:39 AM
Replies: 2
Views: 1,270
Posted By MU Core
Keep in mind you'll require treatment of the...

Keep in mind you'll require treatment of the adapter ends with the USER enzyme to free the circularized ends for downstream sequencing chemistry. This is a step you would have to incorporate into...
Forum: RNA Sequencing 02-20-2017, 07:06 AM
Replies: 1
Views: 1,907
Posted By MU Core
You may find this publication helpful. ...

You may find this publication helpful.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310221/
Forum: Bioinformatics 01-05-2017, 06:50 AM
Replies: 16
Views: 5,127
Posted By MU Core
I think we've figured out the issue with the...

I think we've figured out the issue with the peculiar 3' end plot. Our informatics group noticed that there was no minimum length set for trimming adapter sequence. The adapter sequence starts with...
Forum: Bioinformatics 12-19-2016, 09:42 AM
Replies: 16
Views: 5,127
Posted By MU Core
Brian, you were correct though that these...

Brian, you were correct though that these libraries were not Nextera.
Forum: Bioinformatics 12-19-2016, 09:11 AM
Replies: 16
Views: 5,127
Posted By MU Core
Here are a couple more examples. Sample M is...

Here are a couple more examples. Sample M is that of a DNA PCR-free library sequenced on a HiSeq. Sample W is a TruSeq mRNA library sequenced on a NextSeq.

Brian and Michael's suggestions both...
Forum: Bioinformatics 12-19-2016, 06:15 AM
Replies: 16
Views: 5,127
Posted By MU Core
I observe the same 3' characteristic as...

I observe the same 3' characteristic as previously reported in this thread. A representative plot is provided. The plot shown is a DNA library with insert size >350bp. However, we see this in all...
Forum: RNA Sequencing 10-18-2016, 04:55 AM
Replies: 4
Views: 1,740
Posted By MU Core
Our experience with the Illumina TruSeq kit...

Our experience with the Illumina TruSeq kit suggests a fragmentation of 3-5 minutes is going to be necessary. We achieve an average insert size of 175-200 bp at 3 minutes.
Forum: Illumina/Solexa 07-17-2016, 02:00 PM
Replies: 3
Views: 2,982
Posted By MU Core
Consider the 12 base index used in the following...

Consider the 12 base index used in the following publication.

http://www.ncbi.nlm.nih.gov/pubmed/20534432
Forum: Sample Prep / Library Generation 07-01-2016, 05:12 AM
Replies: 13
Views: 4,565
Posted By MU Core
Consistency can be an issue. Our group performs...

Consistency can be an issue. Our group performs two independent HS Qubit readings per library. Doing so will help catch any outlier measurements due to a pipetting error, etc. The average is then...
Forum: Illumina/Solexa 04-21-2016, 06:48 AM
Replies: 4
Views: 1,375
Posted By MU Core
Our group operates a single HiSeq 2500. We...

Our group operates a single HiSeq 2500. We measure down time as the instrument being unavailable due to a hardware or software issue. Over the past few years it has been <5%. The past 10 months...
Forum: RNA Sequencing 03-16-2016, 05:30 AM
Replies: 10
Views: 3,194
Posted By MU Core
If using bcl2fastq for adapter trimming, I...

If using bcl2fastq for adapter trimming, I believe default minimum-trimmed-read-length is set to 35. If trimming would cut a read down to less than 35 bases then the bases between the end of the...
Forum: Sanger/Dye Terminator 02-08-2016, 06:25 AM
Replies: 2
Views: 6,019
Posted By MU Core
Ghada, you're observing the presence of "dye...

Ghada, you're observing the presence of "dye blobs" which is the result of poor removal of unincorporated dye terminators from the post-sequencing reaction. A common issue in dye terminator...
Forum: RNA Sequencing 01-07-2016, 06:23 AM
Replies: 2
Views: 1,161
Posted By MU Core
From what organism were these samples derived? ...

From what organism were these samples derived? Might is be plastid (i.e. chloroplast) ribosomal RNA?
Forum: Sanger/Dye Terminator 12-30-2015, 05:50 AM
Replies: 3
Views: 5,812
Posted By MU Core
Hello mohd2b, There should be no problem...

Hello mohd2b,

There should be no problem completing the cycles and loading the reaction. We've had a similar issue with a 96 well plate and data looked fine. Good luck.
Forum: Sanger/Dye Terminator 12-29-2015, 06:25 AM
Replies: 3
Views: 5,812
Posted By MU Core
I assume you are using Big Dye v3 chemistry. I...

I assume you are using Big Dye v3 chemistry. I would expect the reaction to work as long as there were no other issues (i.e. primer is annealing, GC-rich template problems, etc.).
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