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Forum: Illumina/Solexa 12-04-2012, 06:54 AM
Replies: 0
Views: 381
Posted By biochembug
Ghost sequences

Hi Friends

Custom protocol sequencing gave 90% junk sequences. Rest is matching the human genome. Majority of junk sequences are repeats of adapter/primer, where the sequencing primer should bind....
Forum: Illumina/Solexa 12-03-2012, 10:48 AM
Replies: 6
Views: 519
Posted By biochembug
Hmm.. So this corroborates the point made by...

Hmm.. So this corroborates the point made by Illumina techsupport. Thanks yaximik.
Forum: Illumina/Solexa 12-03-2012, 06:53 AM
Replies: 6
Views: 519
Posted By biochembug
Here is the expected pattern of the ds product...

Here is the expected pattern of the ds product (*=PS bonds) in the library:

5'Flowcellbinding-----------*N-Insert----------Flowcellbinding3'...
Forum: Illumina/Solexa 12-03-2012, 03:56 AM
Replies: 6
Views: 519
Posted By biochembug
Thanks for the reply, yaximik! I called on...

Thanks for the reply, yaximik!

I called on Illumina techsupport on this question. They say there should not be any problem for PCR products with PS bonds to form clusters. Although they did not...
Forum: Illumina/Solexa 11-30-2012, 12:18 PM
Replies: 6
Views: 519
Posted By biochembug
PS bonds and polymerase

Hi folks

I am planning to use illumina primers RP1 and RPI1 with phosphorothioate bonds between the last base and the penultimate base at the 3' end. So my eventual PCR library will contain two...
Forum: Illumina/Solexa 08-02-2012, 07:13 AM
Replies: 0
Views: 665
Posted By biochembug
low library concentration

Hi Friends

I am using TruSeq whole RNA sequencing kit. I used 50 ng input of earlier purified mRNA and gave a 15 cycle. At the end, the libraries were dissolved in 30 microliter of RSB buffer. Out...
Forum: Illumina/Solexa 07-31-2012, 10:12 AM
Replies: 2
Views: 595
Posted By biochembug
Hi there I am talking about a pGEM-T clone...

Hi there

I am talking about a pGEM-T clone obtained from a Truseq library. When I blast the sequence with Refseq RNA db using BLASTN, it gives no significant hit. However, after BLASTX it had some...
Forum: Illumina/Solexa 07-31-2012, 09:23 AM
Replies: 2
Views: 595
Posted By biochembug
Mitochondrial genome in Tru RNAseq

Hi folks,

What percent of a RNAseq library can be mitochondrial genome? I understand that it should not be there after DNase I treatment. Despite, I have done 2 rounds of oligo dT, I am seeing...
Forum: Illumina/Solexa 07-27-2012, 05:04 AM
Replies: 19
Views: 7,516
Posted By biochembug
Hi folks, How is the following multiplexing...

Hi folks,

How is the following multiplexing (Barcodes 2, 3, 4, 5, 18, 19, 12, 13, 8 libraries) in a lane for TruSeq? In case, it is the problem to pool 2, 3 or 4 libraries I may be safe.

B.No....
Forum: Illumina/Solexa 07-23-2012, 01:40 PM
Replies: 0
Views: 991
Posted By biochembug
barcode sequencing primers

I used the RP1 and RPI1, 2, ..... primer/adapter sequences from the Illumina Customer letter for a custom library. Essentially, these are given for the TruSeq Small RNA sequencing strategy.

I...
Forum: Bioinformatics 05-16-2012, 10:33 AM
Replies: 2
Views: 314
Posted By biochembug
Seems interesting! Thank you.

Seems interesting!

Thank you.
Forum: Bioinformatics 05-16-2012, 05:22 AM
Replies: 2
Views: 314
Posted By biochembug
Reads for a gene

Hi Friends

I am interested to know whether an isoform of a protein (at transcript level) is expressed in different human tissues or cells. My plan is to dig the already available deep sequencing...
Forum: Sample Prep / Library Generation 05-11-2012, 07:40 AM
Replies: 7
Views: 1,070
Posted By biochembug
Hi all, Great to see that you guys are...

Hi all,

Great to see that you guys are keeping this thread alive. As pointed out by Phillip, I am looking for the cut-off of the single stranded DNA molecules on AMPure beads. Comments are always...
Forum: Sample Prep / Library Generation 05-10-2012, 03:40 AM
Replies: 7
Views: 1,070
Posted By biochembug
Thanks for the info Ethan. Will give a shot! ...

Thanks for the info Ethan. Will give a shot!

Biochembug
Forum: Illumina/Solexa 05-10-2012, 03:20 AM
Replies: 2
Views: 655
Posted By biochembug
Thanks Dario. We already thought of a similar...

Thanks Dario. We already thought of a similar CAGE technique exactly from RIKEN labs, however due to the number of steps we were kind of reluctant. Anyhow, I would like to get an answer for my query....
Forum: Illumina/Solexa 05-09-2012, 04:01 PM
Replies: 2
Views: 655
Posted By biochembug
5' end tags

Hi Friends

We are interested in the 5' ends of the transcripts. I was thinking of setting an RNA adapter at the 5' end (as in directional sequencing), then shearing the RNA with Mg+2 at 94degC for...
Forum: Sample Prep / Library Generation 05-09-2012, 01:33 PM
Replies: 7
Views: 1,070
Posted By biochembug
What volume should I use to eliminate low...

What volume should I use to eliminate low molecular weight (<100 ntd) cDNA?
Forum: Sample Prep / Library Generation 05-09-2012, 10:54 AM
Replies: 7
Views: 1,070
Posted By biochembug
AMPureXP for ssDNA

Hi Friends

Has anyone used AMPureXP for ssDNA purification? I need to get rid of smaller cDNAs (<100 ntd).

Biochembug
Forum: Illumina/Solexa 03-02-2012, 09:50 AM
Replies: 3
Views: 1,526
Posted By biochembug
PS=phosphorothioate bonds!

PS=phosphorothioate bonds!
Forum: Illumina/Solexa 02-27-2012, 08:50 AM
Replies: 3
Views: 1,526
Posted By biochembug
Enrichment PCR failure!

Hi Friends

Since a long time now I have been fiddling around some Illumina Truseq primers for a custom sequencing protocol. I am using RP1 and RPI1 (and other indexing primer for my amplification...
Forum: RNA Sequencing 10-09-2011, 03:55 AM
Replies: 6
Views: 1,064
Posted By biochembug
Thanks

Hey that is a good information. I did find the paper. Thank you once again.

Bug
Forum: Illumina/Solexa 09-26-2011, 07:17 PM
Replies: 0
Views: 1,041
Posted By biochembug
sequencing homopolymers

I have few technical queries regarding sequencing some repetitive sequences such as homopolymeric stretches of any base exactly near the start of sequencing primer on Illumina sequencing platform.
...
Forum: Illumina/Solexa 09-22-2011, 05:56 AM
Replies: 1
Views: 810
Posted By biochembug
PolyA on flowcell

Hi Friends

If I plan to clone RNA near its 3' end and somehow select just the 3' ends. What do you think about clusters when sequencing the A tail. I am planning to sequence from the back-end of...
Forum: Illumina/Solexa 09-16-2011, 06:30 PM
Replies: 75
Views: 16,774
Posted By biochembug
Thanks for reply

Hi Friends

Thanks all for posting replies. Some people whom I talked here at my place suggested qPCR can be best. Anyone knows whether some company provides any standard for it? Of course I would...
Forum: Illumina/Solexa 03-04-2011, 09:06 AM
Replies: 0
Views: 884
Posted By biochembug
troubleshooting illumina seq

Dear friends

I have my library which is prepared using custom made primers published in a an article. My eventual product (double stranded of course) will look like this:

...
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