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Thread | Thread Starter | Forum | Replies | Last Post |
trim adapter from Illumina Genome Analyzer IIe miRNA reads | NicoBxl | Bioinformatics | 5 | 01-02-2014 05:31 AM |
Illumina Adapter and Primer preparation | S.Iyengar | Sample Prep / Library Generation | 7 | 11-04-2013 07:08 AM |
primer/adapter sequences | nikiwilson | Sample Prep / Library Generation | 2 | 06-21-2011 01:36 PM |
what's the different between Illumina Genome Analyzer | biocc | Illumina/Solexa | 3 | 06-03-2010 11:28 PM |
A clarification for Illumina/Solexa Genome Analyzer Primer/Adapter Sequences | kaichen | Illumina/Solexa | 1 | 08-06-2009 05:57 PM |
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#41 |
Junior Member
Location: WI Join Date: Sep 2009
Posts: 2
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New to this so my knowledge-base is still quite limited so be gentle in reply, please
![]() We will prepare miRNA-Seq library and send it out for sequencing on a GAII. Can anyone confirm that these adapter/Primer sequences first reported by ECO have (can) been used successfully on the GAII (and presumably GAIIx)? Small RNA oligonucleotide sequences RT Primer 5' CAAGCAGAAGACGGCATACGA 5' RNA Adapter 5' GUUCAGAGUUCUACAGUCCGACGAUC 3' RNA Adapter 5' P-UCGUAUGCCGUCUUCUGCUUGUidT Small RNA PCR Primer 1 5' CAAGCAGAAGACGGCATACGA Small RNA PCR Primer 2 5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA Small RNA Sequencing Primer 5' CGACAGGTTCAGAGTTCTACAGTCCGACGATC Was (is) there a GAI instrument? Does it require different Adapter/Primer sequences? Can't find any evidence of a GAI version on Illumina website. Thanks in advance |
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#42 |
Junior Member
Location: Cambridge Join Date: Jan 2010
Posts: 1
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Hi,
I am New to Illumina sequencing. This is a great resource!!! I was wondering if anyone has figured out the sequences of the multiplexing adaptors. Thanks |
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#43 |
Junior Member
Location: UK Join Date: Feb 2010
Posts: 1
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Thanks for all the helpful stuff on here. A quick newbie question: are all these adapter sequences etc definitely unchanged across the various incarnations of Illumina (GAI, IIe, IIx) - I think so but not certain?
Thanks bp |
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#44 |
Member
Location: California Join Date: Jul 2009
Posts: 46
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i can say the SE adaptors worked on my GAIIx SE sequencing runs last August with the latest kits at the time.
Last edited by der_eiskern; 02-04-2010 at 10:25 AM. Reason: clarification |
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#45 |
Junior Member
Location: Utrecht Join Date: Feb 2010
Posts: 1
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Dear all,
I have 45M (25M in pairs) aligned Solexa reads of 62 bp length in several map files after running Maq map. I have removed the duplicates and now I want to merge them, but get an error message that says that the size of the merged file becomes to big (> 2.1G). How I can get around this? Are there scripts available that allow me to split the mapfiles according to chromosome number? Kind regards Henri Heuven vet.fac.uu |
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#46 |
Junior Member
Location: aberdeen Join Date: Dec 2009
Posts: 5
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Hi
I wonder if somebody can help me here....I have got some solexa data (paired end reads) and want to map these to the genome with bwa/maq...However when I did this only 20% of sequences mapped so I gather I want to check/ for presence of adapters in the sequences and then repeat the alignment after trimming of the data. Which adapter sequences should I screen for- as this is paired end data I assume its the 5` to 3` sequence for the PE adpter 1 and 2 which I found in one of your posts? Paired-end DNA PE Adapter1: 5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3' 3' -------------------- -----TGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGp (-) -------------------- -------------------- -------------------- - 5' PE Adapter2: 5' -------------------- -------------------- ------------------ (-) pGATCGGAAGAGCGGTTCAG CAGGAATGCCGAG------- -------------------- - 3' 3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTC------- -------------------- - 5' In fasta format: >Solexa-PairedEndAdapter1 ACACTCTTTCCCTACACGACGCTCTTCCGATCT >SolexaPairedEndAdapter2 GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG If I had single end solexa data would I then screen for the same adapters Genomic DNA oligonucleotide sequences (from previous posting) Adapters 1 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT Last edited by cur; 03-24-2010 at 03:39 AM. |
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#47 |
Senior Member
Location: Cambridge, MA Join Date: Mar 2009
Posts: 141
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Hi cur,
That is not quite right- you have to use the sequence of the PCR product produced by amplification of your library with paired end primers to identify sequences to screen out. You are looking for sequence between your insert and the flow cell oligo. This would appear in your product only if your insert was incredibly short (or nonexistent)- I suspect you may have sequenced a whole bunch of adapters. What did your Bioanalyzer trace look like? I've found that the molar ratio of amplified adapter sequence (~130 bp) to amplified library is pretty close to the percentage of adapter only reads you get. I believe the following is right but others please correct if not: Sequence between your insert and the flow cell oligo for Round 1 paired end: AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG Sequence between your insert and the flow cell oligo for Round 2 paired end: AGAAAGGGATGTGCTGCGAGAAGGCTAGA For single read the appropriate sequence to screen out depends whether you prepped your libraries with single or paired end adapters. |
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#48 |
Junior Member
Location: San Diego Join Date: Apr 2010
Posts: 1
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Hi,
I purchased Illumina's multiplexing kit and ligated the index PE adaptor to my samples. However, I soon learned that this kit is not compatible with Agilent's SureSelect DNA Capture protocol (microarray based). I would still like to proceed with the library prep (have not added the barcodes/indexes) but was wondering if anyone knew the difference between Illumina's Index PE adaptor and standard PE adaptors. Thanks!! |
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#49 |
Junior Member
Location: Oslo, Norway Join Date: Apr 2010
Posts: 2
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Hi jsilhavy,
the adapters are quite similar because the index is added in the PCR step and does not come with the adapters. I attach a file with the sequences that I found in some other post in this forum. |
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#50 |
Junior Member
Location: aberdeen Join Date: Dec 2009
Posts: 5
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Hi greigite
Thanks for the reply- in this case the answer was actually simple in the end- the sequencing service had not sent us the right data. Once we got the right data there was no problem with the alignment :-) what a relief |
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#51 | |
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Location: uk Join Date: Apr 2010
Posts: 43
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Does anyone know the primer concentrations required for SE ChIP library preparation?
This is the information given in the protocol: Quote:
Thanks. Last edited by AdamB; 04-23-2010 at 07:31 AM. |
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#52 |
Member
Location: uk Join Date: Apr 2010
Posts: 43
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I found the answer in another thread. Just to check, I called Illumina tech support, and they have confirmed the stock PCR primer 1.1 and 2.1 concentration is 25 uM.
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#53 |
Junior Member
Location: harbin,china Join Date: Mar 2010
Posts: 9
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thanks,thats what i want,by the way weather all the illumina use the same adapter?
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#54 |
Junior Member
Location: us Join Date: May 2010
Posts: 5
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Hi all,
Im new to paired end mRNA seq. Are the adapters for the paired end seq same as the genomic DNA adapters? Thanks |
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#55 |
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Location: Sydney, Australia Join Date: Jan 2008
Posts: 83
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@kr067: No. Read the pdf posted by clpX above.
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#56 |
Junior Member
Location: us Join Date: May 2010
Posts: 5
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Thank you sci_guy! that was really helpful!
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#57 |
Member
Location: New York Join Date: Dec 2009
Posts: 17
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I noticed that several people were wondering about adaptor and primer concentrations in the Illumina kit. I just spoke to an Illumina tech support person, and for RNA-seq at least, the working concentration for the adaptor oligo mix is 15 uM while PCR primers are 25 uM.
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#58 |
Junior Member
Location: Spain Join Date: Sep 2009
Posts: 3
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Hi,
I want to do directional mRNA seq. I have to use the Small RNA Sequencing Primer during the primer hybridization. Does anybody know at which concentration should I adjust this primer? thanks |
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#59 |
Member
Location: uk Join Date: Apr 2010
Posts: 43
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I ordered the following primers from Sigma:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T CAAGCAGAAGACGGCATACGAGCTCTTCCGATC*T (HPLC-purified) *=Phosphorothioate bond However I can't get them working with my ChIP input DNA sample. Have tried varying the number of cycles, but each time after the PCR amplification of my library I only have 2 or 3 ng/ul, whereas colleagues have had more like 20 ng/ul. I'm unsure whether the problem is with the primers or something else. As far as I know, everything apart from the primers is the same as colleagues' libraries that worked. I don't have any Illumina primer to test alongside. |
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#60 |
Junior Member
Location: Co Kildare Ireland Join Date: May 2010
Posts: 1
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I'm trying to figure out what the final sequence of an Illumina PE indexed library is and where the enrichment primers and sequencing primers go. Has anyone figured out a schematic of generation of the PE indexed libraries showing all the sequence similar to the one I found on this forum for non-indexed PE libraries (see below).
dandestroy09-18-2008, 07:13 PM Sorry for the small font, but that's the only way I could make it fit PE Adapter1: 5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3' 3' -------------------- -----TGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGp (-) -------------------- -------------------- -------------------- - 5' PE Adapter2: 5' -------------------- -------------------- ------------------ (-) pGATCGGAAGAGCGGTTCAG CAGGAATGCCGAG------- -------------------- - 3' 3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTC------- -------------------- - 5' PE PCR Primer1: 5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3' 3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 5' PE PCR Primer2: 5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 3' 3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGCTAG AGCATACGGCAGAAGACGAA C 5' Result Library: 5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (N) AGATCGGAAGAGCGGTTCAG CAGGAATGCCGAGACCGATC TCGTATGCCGTCTTCTGCTT G 3' 3' TTACTATGCCGCTGGTGGCT CTAGATGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGA (N) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGCTAG AGCATACGGCAGAAGACGAA C 5' PE DNA Sequencing Primer1 5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3' 3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 5' PE DNA Sequencing Primer2 5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 3' 3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGC--- -------------------- - 5' |
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