what is the "molecular indices" in the NEXTflex qRNA kit

[Bioo Scientific]

Hi Kmcarr,

In the set of experiments we describe in the white paper describing the NEXTflex qRNA-Seq Kit our goal was to compare unique fragments determined by reads with unique start and stop sites verses molecular indexing. Figure 5 illustrates under representation of high expressing ERCC and selected mRNAs using start and stop sites as indicators of unique fragments. When molecular indices are used (blue line), the under representation at the high expressing genes is clear. We’re happy to share the individual ERCC RNAs and mRNAs studied if anyone is interested. In this study we didn’t focus on PCR duplicates, in fact we purposely performed the experiment with as few PCR cycles as possible to demonstrate the differences between copy number. We are working on data that describes the benefit of this technology for PCR duplication using Chip-Seq and other applications were PCR cycles are typically higher than they should be.

Cheers,
Dawn

[kmcarr]

The thing I'm not seeing in any of their literature is any actual data on the rate of PCR duplication detected using their technique. With these libraries you can directly measure the rate of PCR duplicate reads, so why haven't they reported how many they find? If PCR duplication is a significant source of error in RNA-Seq data sets why not present data showing that? On the other hand if PCR duplication is generally not a significant factor then there is no reason to use their kit.

Hmm...did I just answer my own question?

[bbeitzel]

I've attached the white paper where they describe the kit. They've commercialized the technique described in 2 of the PNAS papers in the references.

Basically, they have a pool of 96 adapters that have an additional 8-mer barcode downstream of the read 1 and read 2 sequencing primer binding sites. The adapters in the pool are added stochastically to each end of a dsDNA, so you have 96 x 96 = 9216 possible combinations of "molecular barcodes." The barcode combination plus the sequence of the insert allow you to tell if a read is a PCR duplicate (generated during enrichment PCR) or if it is a true duplicate. A PCR duplicate will have the same insert and molecular barcodes. A true duplicate will have the same insert with different molecular barcodes.

The white paper explains it pretty well.

[silin284]

Hi

I just saw the new NEXTflex RNA-Seq kit. They have this interesting "molecular indice" that would label each dsDNA molecule.

http://www.biooscientific.com/DetailProduct.aspx?itemId=496&CName=Next-Generation%20Sequencing&SubName=Illumina-Compatible&Name=RNA%20Sequencing&utm_source=qRNA+Customer+WITHOUT+call+out&utm_campaign=qRNA-Seq+gen&utm_medium=email

When I took a closer look, I can see that this "indice" is most likely to be a pool of barcode adapters (as in Craig et al 2004 Nat Methods). I think they also have the 3' end matches the truseq adapter. Unlike the truseq adapter, one aditional barcode could be placed before the T overhang. Nextflex claims to have 9,216 barcode in each adapter. That is 4x4x4x4x4x3x3. They could synthesize/anneal 9,216 adapters but I think it is more reasonable to use 7 random nucleotide (during oligo synthesis) barcode before the T overhang. In order to make the adapter, they must be able to anneal it to a complementary oligo. I assume they could use one (or a few) with deoxyinosine.

Anyone has more information? I am just guessing, and could be completely wrong.

Cheers
silin