[ShiveringFire]

The indexing strategy should work equally well for paired-end reads (carrying out the index read after the first sequencing reaction, before 'flipping' the product on the PE module and performing the reverse read of the captured sequence, as in the Illumina multiplex protocol), but you would just need to bear in mind that the flow cell oligos C & D on the PE flow cell are slightly longer, so you would need to adjust the adapter/primer tails accordingly.

Exeplex,
I have been staring at your excellent illustration and it led me to generate more questions on Paired-End multiplexing.
During inverse pcr, does the introduction of index sequences to the forward/reverse primers require modification of the PE capture sequencing primers? That is, if one multiplexes 48 samples does s/he have to use 48 different capture sequencing primers? If that is the case, the only solution I could come up with is to shorten the PE sequencing primers into 20-mer and 19-mers so that a single set of primers used (see my illustration attached). However, I am not sure how flexible the illumina chemistry is for this.

[silin284]

I am trying to come up with a library-free way to introduce index (barcode) sequences into circularized captured genome fragments (Molecular Inverted Probes – MIP) that are about 250 nt long. The MIP designs are described in Porreca et al. 2007 and in Turner et al 2009:

http://www.nature.com/nmeth/journal/v4/n11/abs/nmeth1110.html

http://www.nature.com/doifinder/10.1038/nmeth.f.248

The authors ran their 16 samples in separate lanes, therefore did not need to index. The 100-mer oligo MIP designs has a 30 nt common linker sequence, so that one can do inverse pcr using Illumina paired end primers directed against this common linker and then load into flowcell directly for cluster generation.

I like the approach a lot, but couldn’t yet find a way to append index sequences without the standard library prep using adapters described in Cronn et al. or Craig et al. mentioned in this thread.

Any thoughts?

Great idea. Some problem might come from the 30 nt common linker.
1. It could generate bias to DNA with similar sequence (or a particular hairpin structure).
2. It also put a limit on the size of DNA sample.

[Exeplex]

Library-free addition of index sequences in circularized MIP captured sequences?

Hi ShiveringFire,

In my diagram the index is introduced by the reverse primer in the inverse PCR step (called the Capture_Slxa_Rev_Amp primer in the Turner paper), so you would need a unique reverse primer for each index sequence you wanted to use. The forward PCR primer and the sequencing primer are common in all reactions - you need to use a sequencing primer that anneals to the common linker so that this sequence does not end up in your reads, as it would if you used the standard Illumina primer. However, I don't think there's anything special about the chemistry of the sequencing primer, so you can just make a custom one to anneal wherever you need it (as Turner did).

The post above also makes some interesting points, particularly about the insert size - I'm not aware that the MIP strategy has been shown to work on inserts above 120bp, and it's interesting that Jay Shendure's most recent paper made use of arrays rather than MIPs for whole exome sequencing. One other potential problem you might want to consider at an early stage - Agilent no longer seem to make programmable microarrays for the length of oligos you would need, and I'm not aware of an alternative supplier but would welcome any info on this as I need one for a different project!

[ShiveringFire]

Thank you all.

Agilent no longer seem to make programmable microarrays for the length of oligos you would need, and I'm not aware of an alternative supplier but would welcome any info on this as I need one for a different project!

We got our 12.000 oligopool (all 100-mers) synthesized by Combimatrix: http://www.combimatrix.com/ Our MIPs will target inserts less than 190bp. We are expecting 75nt paired-end reads from both ends.

[Corey]

Hi all,
Corey from University of Toronto here. Have followed the great posts here for several months and just wanted to introduce myself. Regarding this multiplex thread, we have recently published a simple 2 step protocol that works great for counting applications. http://genome.cshlp.org/content/19/10/1836.long

[Exeplex]

We got our 12.000 oligopool (all 100-mers) synthesized by Combimatrix: http://www.combimatrix.com/ Our MIPs will target inserts less than 190bp. We are expecting 75nt paired-end reads from both ends.

Thanks Shivering, I'll check them out

[BearClaw]

Great post!

Thanks Exeplex for the great figure. I was halfway through making an almost identical figure for myself when I saw your post. It's nice to see on paper what is going on in my mind!

Hi,

Re. the second part of this post, various groups (e.g. Cronn et al., NAR 2008; Harismendy & Frazer, Biotechniques 2009) have used custom adapters incorporating the barcode so that it becomes part of the read at the 5' end. In a bid to get my own head around the various stages, I've adapted the excellent figure posted by greigite (in the Tech Summary: Illumina's Solexa Sequencing Technology thread) to include some details of the approach used by Cronn et al., as well as a generic strategy to follow through the PCR and sequencing steps (any errors, please let me know!) - hope this is useful.

Also, check out the other posts in this thread for great ideas re. barcode design and bioinformatic processing of sequences.

Exeplex

[epistatic]

Python code for generating Illumina optimized indexes

Hi All,

I was struggling with the best way to get >12-plex indexes as I did not think those in the Craig et al. paper were well balanced to work well with the GAII.

I came across this post and the python script for generating indexes with different parameters, and then a script for picking and evaluating the indexes. The process is well described and I was easily able to run it on my Mac.

Index design and analysis software http://bioinf.eva.mpg.de/multiplex/

Meyer, M., Kircher, M. Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing. Cold Spring Harb. Protoc. (in press) doi: 10.1101/pdb.prot5448.

[greigite]

Hi epistatic,
Thanks for posting the link- very useful! I have tried 6 of the indices in the Craig et al paper and I agree, they are not well balanced for the GAIIx. I definitely saw a reduced cluster density when using the indices as well as the high frequency of skipping the first index base mentioned in the link.

[vasvale]

hello

as some time has past since the last posting I wonder if anybody has an improved method for indexing for single-reads

Vasvale