TruSeq smallRNA libraries

[sajoshi]

I am doing a TruSeq smallRNA library prep. When I was doing Ligate 3' adapter, and came to adding the STP step, the tube in the kit was empty (Illumina glitch). Illumina said to keep samples at -80 and shipped me the STP. Then I continued. When I checked BioA traces (see attached) the band is not where it should be. So it did not work and I will have to repeat. Any suggestions/feedback if you have any experience with this library prep.

[luc]

How did your RNA sample look before the library prep?

[sajoshi]

small RNA library

See attached. Sample was purified when given to me and hence checked on smallRNA chip and BioA conc. was close to the conc. by Qubit microRNA assay. I used a human brain total RNA as control while preparing libraries.

[kerplunk412]

The RNA looks good to me, but the libraries seem to be just adapter-dimer. How much RNA input did you use?

Also, I would like to mention that the TruSeq kit is about the worst Small RNA-Seq kit you can get, at least in terms of cost and data quality. I highly recommend you check out the NEXTflex Small RNA Sequencing Kit v3, as it uses randomized adapters to reduce ligase bias and therefore generate more accurate data and greater discovery/detection rates, it includes completely gel-free or low-input options, and it is much more cost effective.

As a disclaimer, I work for Bioo Scientific.

[sajoshi]

message

The RNA looks good to me, but the libraries seem to be just adapter-dimer. How much RNA input did you use?

Also, I would like to mention that the TruSeq kit is about the worst Small RNA-Seq kit you can get, at least in terms of cost and data quality. I highly recommend you check out the NEXTflex Small RNA Sequencing Kit v3, as it uses randomized adapters to reduce ligase bias and therefore generate more accurate data and greater discovery/detection rates, it includes completely gel-free or low-input options, and it is much more cost effective.

As a disclaimer, I work for Bioo Scientific.

I just send you a message asking a question. Please let me know.

[kerplunk412]

I just send you a message asking a question. Please let me know.

Response sent.

Also, to be fair I should mention some of the other kits out there and the pros and cons.

NEB: Cost effective, lower bias than TruSeq, but substantially more bias than NEXTflex (Bioo)

TriLink: Cost effective (I think), streamlined protocol with no gel size selection required, but similar bias as TruSeq

Clontech: Uses poly(A) tailing and template switching RT to eliminate ligation steps, thereby reducing bias. Shows slightly less bias than NEXTflex kit, but mapping rates to miRNA are very low, leading to lower overall detection/discovery rates in total RNA samples. This makes sense, as other than size selection there is no way to select for miRNA as there is with ligation-based kit. I think this protocol is also pretty streamlined.

[anup.chugani]

Any idea how TriLink works?
They claim to have modified the 3' and 5' adapters.
We have observed the presence of adapter peak at ~60bp. Can it affect the clustering?

[kerplunk412]

TriLink has indeed modified the adapters to greatly reduce adapter-dimer formation, but I am not sure exactly how it works. In my experience their kit works well but still has big issues with bias.

A peak at ~60 bp is most likely excess PCR primer and will not interfere with clustering, as it will not be able to amplify on the flow cell.

[anup.chugani]

Thanks for the reply.
Since it does not cluster, could it occupy the oligoes on the flowcell and stay inert? We have actually observed less cluster numbers for this run.

[kerplunk412]

Unless there is a large amount of excess PCR primer compared to actual library product it should not occupy enough primers on the flow cell to make any difference. The flow cell has enough primers on it to allow single library molecules to make clusters of about 1,000 strands (according to Wikipedia) through bridge amplification, so since the excess PCR primer is not amplified the number of primer sites it occupies on the flow cell will be negligible.