Solutions for multiplexing/barcoding

memento · 09-26-2012, 06:33 AM

Dear All,
Can somebody point me out to a good and reasonably priced barcoding solution for illumina's MiSeq ? Are the adaptor sequences similar between MiSeq/HiSeq ? I found a barcoding generator by comai, but there is no protocol how to use it's output in a lab setting (probably very trivial question). I would assume that this is the cheapest solution. Bioo has a 96samp kit good for 768 reactions (question is how big rx they are talking about?) for 4995$. Are they the cheapest commercial solution ?
Thanks in advance!

TonyBrooks · 10-15-2012, 04:00 AM

What application are you specifically looking at? RNA-Seq, DNA-Seq, ChIP-Seq, other...
What level are you looking to plex (12, 48, 96, higher?)
IMHO indexing primers are the most over-priced aspect of NGS. If you think you are going to be doing a lot of index library preparation, you may want to look at making your own.

http://www.ncbi.nlm.nih.gov/pubmed/21431778

We ordered a set of indexes based on the above paper and use them regularly for both indexed (and non-indexed runs - you don't need to read the barcode). It works to be significantly cheaper per reaction than buying commercially, but there is obviously a largish initial outlay on the HPLC oligos. (Of course, you don't need to order all 96 in one go - one synthesis should provide enough for hundreds of reactions).
Those oligo sequences work well on the GAIIx, HiSeq and MiSeq - just remember to use the custom index sequencing primer.

There are also people on here dabbling with homebrew dual indexing, which is something we may look at in the future. It cuts the number of oligos you need to order down and will hopefully be scalable to >96 plex.

JackieBadger · 10-15-2012, 04:23 AM

for MiSeq amplicon work we use the 454 MIDs

bhennuy · 12-05-2012, 05:13 AM

Hello,
We are interested in using dual indexing strategy for multiplexing more than 384 samples in one run on MiSeq instrument. Could we use the 48 indexes (6 bases) provided by Illumina for Truseq Small RNA sample prep as i7 indexes ? how to add 2 bases to these sequences to get unique 48 indexes ? We would use 8 i5 (as Nextera) indexes to reach 384 unique combinations. Do you think this strategy would work? Could we use more than 8 i5 indexes ?
Thans for your help.

TonyBrooks · 12-05-2012, 05:25 AM

You could look at this paper from Sanger
http://www.ncbi.nlm.nih.gov/pubmed/21431778
They use 96-plex 8-mer barcodes. You could use those sequences for your i7 indexes.

bhennuy · 12-05-2012, 07:43 AM

Thank you TonyBrooks for the info, I will read it carefully. Do you know why Illumina do not provide more i5 indexes to increase the number of multiplexes ?

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09-26-2012, 06:33 AM	#1
memento Member Location: UK Join Date: Nov 2010 Posts: 49	Solutions for multiplexing/barcoding Dear All, Can somebody point me out to a good and reasonably priced barcoding solution for illumina's MiSeq ? Are the adaptor sequences similar between MiSeq/HiSeq ? I found a barcoding generator by comai, but there is no protocol how to use it's output in a lab setting (probably very trivial question). I would assume that this is the cheapest solution. Bioo has a 96samp kit good for 768 reactions (question is how big rx they are talking about?) for 4995$. Are they the cheapest commercial solution ? Thanks in advance!

10-15-2012, 04:00 AM	#2
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	What application are you specifically looking at? RNA-Seq, DNA-Seq, ChIP-Seq, other... What level are you looking to plex (12, 48, 96, higher?) IMHO indexing primers are the most over-priced aspect of NGS. If you think you are going to be doing a lot of index library preparation, you may want to look at making your own. http://www.ncbi.nlm.nih.gov/pubmed/21431778 We ordered a set of indexes based on the above paper and use them regularly for both indexed (and non-indexed runs - you don't need to read the barcode). It works to be significantly cheaper per reaction than buying commercially, but there is obviously a largish initial outlay on the HPLC oligos. (Of course, you don't need to order all 96 in one go - one synthesis should provide enough for hundreds of reactions). Those oligo sequences work well on the GAIIx, HiSeq and MiSeq - just remember to use the custom index sequencing primer. There are also people on here dabbling with homebrew dual indexing, which is something we may look at in the future. It cuts the number of oligos you need to order down and will hopefully be scalable to >96 plex. Last edited by TonyBrooks; 10-15-2012 at 04:03 AM.

12-05-2012, 05:13 AM	#4
bhennuy Junior Member Location: Belgium Join Date: May 2009 Posts: 6	indexes sequence for multiplexed > 96 Hello, We are interested in using dual indexing strategy for multiplexing more than 384 samples in one run on MiSeq instrument. Could we use the 48 indexes (6 bases) provided by Illumina for Truseq Small RNA sample prep as i7 indexes ? how to add 2 bases to these sequences to get unique 48 indexes ? We would use 8 i5 (as Nextera) indexes to reach 384 unique combinations. Do you think this strategy would work? Could we use more than 8 i5 indexes ? Thans for your help.

10-15-2012, 04:23 AM	#3
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 380	for MiSeq amplicon work we use the 454 MIDs

12-05-2012, 05:25 AM	#5
TonyBrooks Senior Member Location: London Join Date: Jun 2009 Posts: 298	You could look at this paper from Sanger http://www.ncbi.nlm.nih.gov/pubmed/21431778 They use 96-plex 8-mer barcodes. You could use those sequences for your i7 indexes.

12-05-2012, 07:43 AM	#6
bhennuy Junior Member Location: Belgium Join Date: May 2009 Posts: 6	Thank you TonyBrooks for the info, I will read it carefully. Do you know why Illumina do not provide more i5 indexes to increase the number of multiplexes ?