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Forum: Sample Prep / Library Generation 08-17-2012, 11:45 AM
Replies: 0
Views: 1,024
Posted By monad
Library automation with E220 and Sciclone

We are considering Covaris E220 (either parallel or serial) and Caliper (PerkinElmer) Sciclone to improve NGS library prep automation.
Any suggestion to look for better alternative or comments on...
Forum: Core Facilities 06-29-2011, 10:40 PM
Replies: 7
Views: 4,520
Posted By monad
Hi megalodon, As you know that the main...

Hi megalodon,

As you know that the main reason we want to do qPCR was to measure the quantity of intact library, so we can maximize the output. I have seen enough that amplification efficiency of...
Forum: Core Facilities 06-15-2011, 02:13 PM
Replies: 9
Views: 4,579
Posted By monad
We can get as high as 130M reads per lane of...

We can get as high as 130M reads per lane of HiSeq. However, your target should be more close to 100M passing reads (or not to exceed much beyond 100M) as HiSeq is more sensitive to high density...
Forum: Genomic Resequencing 05-21-2011, 09:48 PM
Replies: 2
Views: 2,080
Posted By monad
GSNAP/GMAP works very well for us. Here is the...

GSNAP/GMAP works very well for us. Here is the link.
http://research-pub.gene.com/gmap/
Forum: Illumina/Solexa 05-21-2011, 01:02 PM
Replies: 8
Views: 3,371
Posted By monad
long insert mate pair is nightmare with all sort...

long insert mate pair is nightmare with all sort of problem you guys already discussed. Large amount of starting material, overnight gel separation on LMT agarose, quality check for adapter dimers,...
Forum: RNA Sequencing 05-19-2011, 08:37 PM
Replies: 24
Views: 14,407
Posted By monad
DSN works for sub pg level of input RNA (not...

DSN works for sub pg level of input RNA (not detectable with Qubit). total RNA was from single cell layer of insect brain and impossible to secure enough total RNA for 500pg range. We got 30M passing...
Forum: Illumina/Solexa 05-19-2011, 08:27 PM
Replies: 4
Views: 3,163
Posted By monad
We've made more than 1200 library since early...

We've made more than 1200 library since early this year using TruSeq. Very robust and good quality for Illumina sequencing. Much simplified compared to prior version of library kit.
Forum: Vendor Forum 05-18-2011, 09:48 PM
Replies: 10
Views: 7,181
Posted By monad
1 ug of input usually good for the Illumina prep....

1 ug of input usually good for the Illumina prep. Do not know about the Nextflex.
Forum: Sample Prep / Library Generation 05-18-2011, 01:38 PM
Replies: 4
Views: 4,241
Posted By monad
Make sure that your starting input DNA is enough....

Make sure that your starting input DNA is enough. Longer the insert, you will need a lot more input DNA. We are talking about ~10ug for end repair for ~12kb insert mate pair, so starting material is...
Forum: RNA Sequencing 05-18-2011, 01:35 PM
Replies: 7
Views: 2,681
Posted By monad
We worked with more than 8,000 library prep until...

We worked with more than 8,000 library prep until today, so we are not inexperienced users here for Illumina library prep.

However, DSN is not the way to go if you want to remove ribosomal RNA!
...
Forum: Core Facilities 05-12-2011, 10:51 AM
Replies: 7
Views: 4,520
Posted By monad
qPCR is essential if you want to maintain...

qPCR is essential if you want to maintain expected range of output. Make sure you include standard library that was prepared using same protocol for your library to be qPCR'ed and that was already...
Forum: Vendor Forum 05-12-2011, 10:41 AM
Replies: 10
Views: 7,181
Posted By monad
Excellent!

Excellent!
Forum: Illumina/Solexa 05-12-2011, 10:36 AM
Replies: 17
Views: 5,827
Posted By monad
I guess we are talking about two different things...

I guess we are talking about two different things here. Criteria for the successful sequencing from the instrument operators standpoint vs data content. Not knowing what is the nature of your input...
Forum: Illumina/Solexa 05-12-2011, 10:24 AM
Replies: 17
Views: 5,827
Posted By monad
That is 80M x 200 (2x100), so 16G per lane. ...

That is 80M x 200 (2x100), so 16G per lane.
Again, you need to revisit the goal of your sequencing.
Your data may have lots of room to tolerate with marginal quality or not depending on what you'd...
Forum: Illumina/Solexa 05-12-2011, 08:09 AM
Replies: 17
Views: 5,827
Posted By monad
You have very noisy quality score from the...

You have very noisy quality score from the beginning of sequencing. What was the materials you sequenced? Any chance this is reduced genome or amplicons? Whether passing filtered portion of data is...
Forum: The Pipeline 05-11-2011, 04:08 PM
Replies: 4
Views: 4,985
Posted By monad
I'd like to be wrong, but this may not be in a...

I'd like to be wrong, but this may not be in a prime time within 2-3 years.
Forum: Vendor Forum 05-11-2011, 04:06 PM
Replies: 10
Views: 7,181
Posted By monad
I would appreciate to learn about the...

I would appreciate to learn about the availability of strand strand specific (directional mRNA) kit from your company and whether that is compatible with Illumina TruSeq sequencing. Thanks!
Forum: Pacific Biosciences 05-11-2011, 04:00 PM
Replies: 1
Views: 2,760
Posted By monad
Instrument operation is literally ~10 min. Very...

Instrument operation is literally ~10 min. Very straight forward. If you think that Illumina library prep is reasonable enough, PacBio library prep is very straight forward and equally easy.
Forum: Illumina/Solexa 05-11-2011, 03:55 PM
Replies: 2
Views: 3,294
Posted By monad
Through unofficial source, I was told that...

Through unofficial source, I was told that ChIP-TruSeq will be available soon with barcode.
Forum: Illumina/Solexa 05-09-2011, 09:26 PM
Replies: 14
Views: 4,280
Posted By monad
Today's $5k per genome is based on improved...

Today's $5k per genome is based on improved chemistry for 600Gb. This output seems based on 16 channels (two entire flowcell) at 2x100 at ~190M cluster density. My realistic expectation based on...
Forum: RNA Sequencing 05-09-2011, 04:47 PM
Replies: 2
Views: 4,765
Posted By monad
DSN protocol really works. We used non detectable...

DSN protocol really works. We used non detectable amount of total RNA as an input for library prep and got ~30M passing reads from single GAIIx lane. Unique read was ~5M, so higher redundancy as you...
Forum: Core Facilities 05-08-2011, 05:23 PM
Replies: 9
Views: 4,579
Posted By monad
try newly organized genome center, global genome...

try newly organized genome center, global genome research institute, www.ggri.org

Pricing is one thing. They will consult with you first to figue out the most efficient experimental design and...
Forum: Illumina/Solexa 05-08-2011, 10:15 AM
Replies: 74
Views: 34,966
Posted By monad
2nd gel purification is the best option here, but...

2nd gel purification is the best option here, but you lose lots of library DNA. In this case, you can set additional round of PCR will take care of it. Stick with not more than 5 round of PCR. If...
Forum: Illumina/Solexa 05-08-2011, 09:43 AM
Replies: 14
Views: 4,280
Posted By monad
Another way to look at the HiSeq output is...

Another way to look at the HiSeq output is 80-100M passing reads per lane. You can multiply with your sequencing reads to get your Gb. For Example, with 2x100 you can expect about >16Gb output if you...
Forum: Service Providers 05-08-2011, 09:32 AM
Replies: 11
Views: 4,180
Posted By monad
Consider Global Genome Research Institute

Newly formed non-profit, working with number of genome centers to get the job done. May not be cheapest, but one of high quality data producer. www.ggri.org
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