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Forum: Pacific Biosciences 04-27-2018, 12:59 AM
Replies: 0
Views: 1,197
Posted By weigrc
Set target P1 value at >50%, with the lastest Sequel reagents and SMRT Link v5.1

Hello all,

Regarding P1 value from Sequel runs, we had been told that at <40% is good while P0 and P2 share the rest evenly.

But refer to the attached, the technical note now is saying to set...
Forum: Pacific Biosciences 09-20-2017, 01:20 AM
Replies: 1
Views: 1,521
Posted By weigrc
Shotgun metagenomics study for environmental samples using PacBio

Dear all,

Anyone has such experience? How to conquer the issue of low input DNA?


Thanks,
Wei :confused:
Forum: Pacific Biosciences 09-19-2017, 11:29 PM
Replies: 7
Views: 2,152
Posted By weigrc
We used diffusion loading to keep every size of...

We used diffusion loading to keep every size of transcript for sequencing. And refer to below for the versions.

Sequel Sequencing Kit: V2
ICS SW: V5.0.0
SMRT Link: V5.0.1

Thanks,
Wei
Forum: Pacific Biosciences 09-19-2017, 01:44 AM
Replies: 7
Views: 2,152
Posted By weigrc
Cool Dear all, The attached is what we recently...

Dear all,

The attached is what we recently done for Iso-Seq application.

We are first time to spike-in Sequel SMRT Cell Control Complex 2.0, anyone knows how to interpret its sequencing data?
...
Forum: Pacific Biosciences 06-26-2017, 05:28 PM
Replies: 10
Views: 3,025
Posted By weigrc
Hi Jharting, Thank you for these...

Hi Jharting,

Thank you for these information, and I am reading the documents, they seem useful. thanks!
Forum: Pacific Biosciences 06-23-2017, 05:20 PM
Replies: 2
Views: 2,050
Posted By weigrc
Any schedules to have such Meeting/Workshops in...

Any schedules to have such Meeting/Workshops in Asia?

Thanks,
Wei
Forum: Pacific Biosciences 06-23-2017, 05:15 PM
Replies: 5
Views: 2,709
Posted By weigrc
SMRT cell delivery schedule is ok, but its...

SMRT cell delivery schedule is ok, but its shelf-life, usually 1-2 months left after arrived. And although SMRT cells is 4 per tray for purchase, but the sequencing kit is one plate for 8 cells,...
Forum: Pacific Biosciences 06-23-2017, 05:00 PM
Replies: 10
Views: 3,025
Posted By weigrc
Thank you, jharting. Do you know what...

Thank you, jharting.

Do you know what algorithm SMRT Link is using for demultiplexing? I mean it requests CCS read or full-pass subread? Is it possible to access such setting?

The barcoded WGS...
Forum: Pacific Biosciences 06-22-2017, 06:51 PM
Replies: 10
Views: 3,025
Posted By weigrc
We were using PacBio SMRTbell Barcoded Adapter...

We were using PacBio SMRTbell Barcoded Adapter Complete Prep Kit-96.

And we did contact our tech support to follow up this issue since this Tuesday with the full information they needed, but the...
Forum: Pacific Biosciences 06-21-2017, 10:27 PM
Replies: 10
Views: 3,025
Posted By weigrc
Problem of demultiplexing 4 barcoded E.coli genomes in one Sequel SMRT cell

Dear all,

As captioned, this sequencing included 4 barcoded libraries prepared by the following protocol, and barcode lbc01, lbc02, lbc03 and lbc04 were chosen, respectively. The library size is...
Forum: Pacific Biosciences 06-19-2017, 12:33 AM
Replies: 2
Views: 1,716
Posted By weigrc
With PacBio 10kb protocol, we got the library...

With PacBio 10kb protocol, we got the library sizing around 7kb on agarose gel, no need to do size selection.
Forum: Epigenetics 09-16-2015, 05:52 PM
Replies: 4
Views: 3,573
Posted By weigrc
Dear Nucacidhunter and Avilella, Thanks for...

Dear Nucacidhunter and Avilella,

Thanks for your information. They help!!

BTW, rather than EpiGenome and TruSeq DNA Methylation kits, I read there is a new kit for methylation study as...
Forum: Epigenetics 09-07-2015, 08:02 PM
Replies: 4
Views: 3,573
Posted By weigrc
Duplication rate for whole genome bisulfite sequencing

Hi folks,

For WGBS, we've experienced with human gDNA using Illumina EpiGnome/TruSeq DNA Methylation kit and HiSeq sequencing.

The sequencing duplication rate is dynamic, from <5% to >20%; the...
Forum: Illumina/Solexa 05-03-2015, 06:30 PM
Replies: 4
Views: 1,133
Posted By weigrc
This project is for Reduced Representation...

This project is for Reduced Representation Bisulfite Sequencing (RRBS).

I was thinking it should be no problem with combining RR PE50 and the HO PE50 (derived from HO PE100) data. But Illumina...
Forum: Illumina/Solexa 04-28-2015, 11:31 PM
Replies: 4
Views: 1,133
Posted By weigrc
Just HO PE100 sequencing is the most common run...

Just HO PE100 sequencing is the most common run type here, more cost effective as well. We need to get more sequencing reads for downstream data analysis. Thinking if there is any Bioinformatics...
Forum: Illumina/Solexa 04-27-2015, 11:16 PM
Replies: 4
Views: 1,133
Posted By weigrc
Is it ok to combine RR run and HO run data for downstream data analysis?

Dear all

One of our projects needs PE50 sequencing.

With Illumine HiSeq 1500, one Rapid Run PE50 has been accomplished, but per sample sequencing data (~60% of total) is not sufficient. We are...
Forum: Illumina/Solexa 01-19-2015, 04:46 PM
Replies: 6
Views: 2,789
Posted By weigrc
Info can be found as below. Briefly, V2 generates...

Info can be found as below. Briefly, V2 generates the same sequencing output and quality as V1 does, but V2 can have PE250 run.
...
Forum: Sample Prep / Library Generation 12-22-2014, 04:38 PM
Replies: 2
Views: 907
Posted By weigrc
Dear Cliffbeall, Thanks for the idea, yes,...

Dear Cliffbeall,

Thanks for the idea, yes, it can be such case. And anyone has a similar phenomena can share with? I am a bit worry about if the extra peak is a contamination that will affect the...
Forum: Sample Prep / Library Generation 12-21-2014, 06:38 PM
Replies: 2
Views: 907
Posted By weigrc
An extra RNA peak positioned between 16s and 23s RNAs, what it could be??

Hello everyone,

As captioned, 2 (#01 and #03) and 2 (#06 and #09) RNAs (refer to attached) were harvested from the same E.coli in growth phase and post-growth phase, respectively.

A strange RNA...
Forum: Illumina/Solexa 11-30-2014, 05:03 PM
Replies: 6
Views: 2,789
Posted By weigrc
We were going to try RR V2 reagents, but paused...

We were going to try RR V2 reagents, but paused the move after we knew it needs to have a software update + maintenance wash chemical changed first...
Forum: Illumina/Solexa 10-08-2014, 08:13 PM
Replies: 5
Views: 2,210
Posted By weigrc
It is normal to have less than 40% of input DNA...

It is normal to have less than 40% of input DNA recovered as adapter-ligated molecules; for those DNA who don't have a pair of adapters, can not be sequenced. Using QPCR for calculating the library...
Forum: Illumina/Solexa 09-17-2014, 10:04 PM
Replies: 25
Views: 3,189
Posted By weigrc
After these FastQC reports and your explanation,...

After these FastQC reports and your explanation, the issue should not be generated by over-clustering.

As nucacidhunter said, you may give more info for trouble shoot, like listed below:

(1)...
Forum: Illumina/Solexa 09-16-2014, 01:38 AM
Replies: 25
Views: 3,189
Posted By weigrc
If the libraries are ~380 bp, the sequencing...

If the libraries are ~380 bp, the sequencing quality issue should be caused by cluster over saturation.

What are the values of Cluster Density (K/mm2) and Cluster PF (%)???

Though Read 1 and...
Forum: Illumina/Solexa 09-09-2014, 08:22 PM
Replies: 1
Views: 676
Posted By weigrc
We did try this combination before, and it worked...

We did try this combination before, and it worked well. Just to take small sequencing proportion for miRNA libraries, because it is easy to read into adapter sequence then lower sequencing quality...
Forum: Illumina/Solexa 08-05-2014, 10:22 PM
Replies: 2
Views: 1,110
Posted By weigrc
Hi Chipgal, We see the adapter dimers...

Hi Chipgal,

We see the adapter dimers sometimes. Usually do 1X AMPure beads purification to get rid of the dimers, as it affects the number of sequencing reads a lot.

Thanks,
Wei
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