[carmeyeii]

Hi pmiguel

Can you expand on this topic a bit?

Thanks,
Carmen

[pmiguel]

Hi pmiguel

Can you expand on this topic a bit?

Thanks,
Carmen

Sure. In "High output mode", (aka v3 flowcell) the attendant primer mixes are not sufficient to prime all the Dual Barcode reads. Particularly the i5 index read. MiSeq and HiSeq using Rapid Chemistry Cluster kit do include this primer.

So if you want the 50% more reads available from a High Output run, you would need to pay for a "TruSeq Dual Index Sequencing Primer Box" (Lists for around $86.) Also you would need to pay for some extra cycles of SBS potentially. The normal 200 cycle SBS kit can only do about 209 cycles. That is enough to do 2x101 reads + a 7 base index read. But not enough to do a 2x101 + 2x8 index reads. So you either have to buy 4 50 cycle SBS kits for that, or if you were doing both flowcells, it might be cheaper to buy two 200 cycle SBS kits (one for each flowcell) plus a single 50 cycle kit to split between the two. The first option would cost about $700-$800 more.

If you are running a full flowcell of dual index libraries, it doesn't add all that much to the cost per lane. Maybe $100/lane in extra reagents. But obviously if only one lane is going to use dual index libraries you would be paying a large premium. In which case you might be better off just using Rapid Chemistry, since that has overages and primers built into it to allow for dual index libraries.

--
Phillip

[kcchan]

The dual index primer box is only required for Nextera libraries or dual indexing on single read flow cells. You can sequence TruSeq HT with dual indexing on paired end flow cells with just the standard set of index primers (HP8) and 7 extra cycles of sequencing reagents. This is because index 2 uses a neat trick where the P5 adapter on the flow cell serves as the primer for index 2. This is also why the 7 "dark cycles" are needed; to get past the padding region of the adapter sequence.

If you're using a single read flow cell, then you will need to buy the dual index primer kit. The box contains the second primer (HP9) needed to read index 2 without the paired end primers.

Source:

[arcolombo698]

Trimming adapters

Hello.

When trimming the adapters, it is correct to just specify the correct sequences corresponding to the index used ?

why not just use the universal adapter?

thank you

[GenoMax]

Hello.

When trimming the adapters, it is correct to just specify the correct sequences corresponding to the index used ?

why not just use the universal adapter?

thank you

Indexes for illumina are read independently and are never part of the actual sequence. You do not need to use index sequences when trimming data.

[arcolombo698]

Thank you, this answers my question.

for trimming my data, I made sure to include the Universal adapter and the full Adapter Index Sequence (that contains the index), and not just the 6 nucleotide index itself, these get attached after the fragment and need to be trimmed.

thank you