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Forum: Sample Prep / Library Generation 02-23-2018, 07:22 AM
Replies: 5
Views: 2,323
Posted By melop
We just follow the DIY SPRI bead protocol here...

We just follow the DIY SPRI bead protocol here https://openwetware.org/wiki/SPRI_bead_mix. We tested our home-brewed mixture with Ampure beads and didn't find much difference.

It is extremely...
Forum: Sample Prep / Library Generation 01-17-2018, 07:08 AM
Replies: 14
Views: 7,027
Posted By melop
I elute in 30ul of Tris pH=8.0. I pipette it...

I elute in 30ul of Tris pH=8.0. I pipette it until the beads get re-suspended, and directly used that mixture for down-stream workflows without removing the beads.

If you want to remove the beads,...
Forum: Sample Prep / Library Generation 01-16-2018, 03:48 PM
Replies: 14
Views: 7,027
Posted By melop
You can do as low as 0.6X beads. I usually use...

You can do as low as 0.6X beads. I usually use 1X. It usually doesn't matter too much for genomic extracts. I also keep the beads in, they don't affect with PCR or library prep.
Forum: Sample Prep / Library Generation 08-19-2016, 09:33 AM
Replies: 14
Views: 7,027
Posted By melop
Yes - I agree. Didn't add tween 20 so recovery is...

Yes - I agree. Didn't add tween 20 so recovery is probably lower than optimal, but it's more than enough for what I need so can't complain. Attached is a gel picture of genomic DNA extracted directly...
Forum: Sample Prep / Library Generation 08-19-2016, 06:03 AM
Replies: 14
Views: 7,027
Posted By melop
It works for genomic DNA. I now use SPRI beads...

It works for genomic DNA. I now use SPRI beads for extracting genomic DNA either directly from proK-SDS digested tissue or from the supernatant cleaned by phenol-chloroform.
Forum: Sample Prep / Library Generation 07-13-2016, 11:49 PM
Replies: 10
Views: 1,443
Posted By melop
Thanks! I will try cleaning with 1X beads then to...

Thanks! I will try cleaning with 1X beads then to see if the size becomes expected.

Another question though is the double peaks after ligation. From previous posts some seems to suggests that the...
Forum: Sample Prep / Library Generation 07-13-2016, 10:29 PM
Replies: 10
Views: 1,443
Posted By melop
Right, so a PCR clean up won't help much with...

Right, so a PCR clean up won't help much with shifting the distribution up (to peak at 450bp )?
Forum: Sample Prep / Library Generation 07-13-2016, 03:17 PM
Replies: 10
Views: 1,443
Posted By melop
Haven't done the cleanup yet because in the...

Haven't done the cleanup yet because in the protocol I am using it asks for a double cut bead selection, after which the distribution will differ from the raw pcr. If I use a 1.8x clean up the peak...
Forum: Sample Prep / Library Generation 07-13-2016, 09:47 AM
Replies: 10
Views: 1,443
Posted By melop
Today I performed more tests. I adjusted the...

Today I performed more tests. I adjusted the fragmentation step to get a bigger gDNA distribution. I also left the beads in after ligation clean-up for PCR. This seems to help with the size...
Forum: Sample Prep / Library Generation 07-12-2016, 11:08 PM
Replies: 10
Views: 1,443
Posted By melop
Dear Adam, thank you for the suggestions. I...

Dear Adam, thank you for the suggestions.

I always get a peak at around 303bp after PCR, regardless of what size selection I tried before PCR. The first figure shows the fragmented DNA (without...
Forum: Sample Prep / Library Generation 07-12-2016, 06:35 AM
Replies: 10
Views: 1,443
Posted By melop
Always get the same insert size after PCR despite initial size selection on gDNA

Dear All, I am trying to make our own WGS library with dual barcodes. It involves a very common workflow including fragmentation (we use a Shearase from Zymo instead of covaris), End-Repair +...
Forum: Bioinformatics 02-22-2016, 02:14 AM
Replies: 1
Views: 3,628
Posted By melop
This is caused by your system preventing a hard...

This is caused by your system preventing a hard link using C's link() function.

Change psx.c to allow an explicit copying is a workaround:
Forum: Illumina/Solexa 01-27-2016, 10:20 AM
Replies: 33
Views: 5,353
Posted By melop
Yes, they said they will rerun if illumina...

Yes, they said they will rerun if illumina replaces the reagent for them. They did not say what happens if not. But from the message they seemed to imply that rerunning is contingent upon their...
Forum: Illumina/Solexa 01-27-2016, 09:15 AM
Replies: 33
Views: 5,353
Posted By melop
We are puzzled too. Last time I heard from them...

We are puzzled too. Last time I heard from them the core told us that they will only replace the run if Illumina replaces reagents for them. They were vague about what to do if Illumina doesn't. That...
Forum: Illumina/Solexa 01-27-2016, 09:07 AM
Replies: 33
Views: 5,353
Posted By melop
So I have 2 lanes, both of which fall below 75%...

So I have 2 lanes, both of which fall below 75% >Q30 (60% and 70%). As far as I know, the other 6 lanes are all borderline, right at 75% >Q30. So the average across the whole flowcell would still be...
Forum: Illumina/Solexa 01-27-2016, 09:00 AM
Replies: 33
Views: 5,353
Posted By melop
This looks interesting and definitely will be...

This looks interesting and definitely will be useful for downstream analyses.
However I feel that the statistics should be measured in the same way Illumina does, since Illumina guarantees 75%...
Forum: Illumina/Solexa 01-19-2016, 03:13 PM
Replies: 33
Views: 5,353
Posted By melop
I see. But is that a common practice to let the...

I see. But is that a common practice to let the end user bare the loss even if the facility prepares the library knowing that they are going on a HiSeq 4000? I understand that if Illumina agrees to...
Forum: Illumina/Solexa 01-19-2016, 02:16 PM
Replies: 33
Views: 5,353
Posted By melop
Dear all. I'm an end user. Recently we got 2...

Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad,...
Forum: Bioinformatics 10-05-2012, 08:17 AM
Replies: 8
Views: 5,615
Posted By melop
Unfortunately I haven't found a solution for it....

Unfortunately I haven't found a solution for it. Perhaps try remaking the bam files would help?
Forum: Bioinformatics 06-02-2012, 10:32 PM
Replies: 8
Views: 5,615
Posted By melop
I'm extremely annoyed by this problem too. The...

I'm extremely annoyed by this problem too.
The problem seems to have been caused by bcftools when it's trying to convert the uncompressed bcf output from samtools mpileup. This might be caused by...
Forum: De novo discovery 06-01-2012, 02:31 PM
Replies: 1
Views: 1,373
Posted By melop
I have exactly the same question. How to generate...

I have exactly the same question. How to generate the contig link file by aligning the contigs to the ref with, say, nucmer?
Forum: De novo discovery 06-01-2012, 11:51 AM
Replies: 1
Views: 1,261
Posted By melop
Hi There, I want to do the same thing...

Hi There,

I want to do the same thing too. We have some PE and SE illumina reads (30x total), a pretty fragmented genome from a closely related species and a 454 as well as a PE RNA-seq...
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