Library information needed for loading onto sequencer

[MKD]

Hi,

I was looking for some information from anyone running a core or sequencing service. If I had a prepped library, what information about it will you need before you load onto the sequencer from the customer? If it is just total RNA- what is the information you will need for library prep and sequencing.

Thank you.

[nucacidhunter]

1- Kit used for library prep
2- Deviation from supplier recommended protocol
3- Species or tissue
4- Library index

[GenoMax]

Though most facilities will do their own QC (concentration, approx insert size) you could provide them with what you think are values for those two.

[MKD]

Thank you nuacidhunter and Genomax for your replies. I have never used a sequencer myself so am a novice here. I am trying to understand the necessary library parameters to be known when you load onto one lane vs libraries that go on different lanes. Are they the same? For example- I have 50 libraries that I want to load onto the same lane of a Hiseq so what parameters for them have to be same. I am assuming- size, conc, index, sequencing primers, made with the same kit, qc/pooled. Now we have a different set of 40 libraries that need to go onto another lane of the same sequencer for a sequencing run. What parameters need to be same here vs the different lane.
I am trying to come up with a generalized information that would apply for a sequencing run for customers.
Thank you.

[nucacidhunter]

You can pool any library in one lane or flow cell considering following:

1- Pool libraries in pM ratios based on required output, they do not have to be equimolar
2- Libraries pooled in one lane should have compatible (color balanced) indices and good distance in sequence to avoid miss-assignments of reads
3- Libraries with larger fragment sizes will have less read output if pooled with shorter libraries
4- Required sequencing primers for R2 and index reads should be the same for all libraries in one flow cell. Read 1 sequencing primers can be different for different lanes.

[MKD]

Just to summarize then for a library to go on a sequencer for a run, the following information is needed-

1. type of sequencer, -- type of RUN mode? rapid vs normal
2. SR-PE flowcell (has to be either or right ? cannot have 2 types on there)
3. library info- conc, vol, Q/C, pooling info, (why is library type important for sequencer)
4. Sequencing primers- R1, R2, Index OR CUSTOM?
5. Is cycles important? R1, R2 cycles and Index 1 and 2 cycles.
6. Anything else I am missing here.

Thanks a bunch for info.

[GenoMax]

You have not told us what kind of an experiment this is since the library insert sizes needed may differ based on that. Choice of sequencer/run may ultimately depend on cost and type of data you want to get from the experiment (length/SE or PE).

[MKD]

You have not told us what kind of an experiment this is since the library insert sizes needed may differ based on that. Choice of sequencer/run may ultimately depend on cost and type of data you want to get from the experiment (length/SE or PE).

Hi Genomax,

Its not experiment-specific.I was trying to work out a general guideline for a novice to choose their sequencing options given they have their libraries ready to go. So someone comes with prepped libraries- and wants to use a sequencing service. What options would they get starting from the type of instrument. Lets assume we are using illumina platform only.
Right now, I am working with microbial samples that have small genomes so I could do multiple samples per lane. For a set of libraries to be put on a sequencer- what are the minimum parameters we need to specify assuming a full flowcell in Nextseq or a few lanes in Hiseq. The question about multiple lanes and library requirements for being loaded into adjacent lanes is for the Hiseq option.

[GenoMax]

https://genohub.com/ and http://allseq.com/ are sequencing marketplaces that provide comparative shopping for NGS services. They also have knowledge banks. You may want to create an account and walk through their project setup to get answers for some of the questions you are thinking about.

[MKD]

Thanks Genomax. Actually, I am in contact with Genohub already. I will check out their site in more detail.

[kmcarr]

You can pool any library in one lane or flow cell considering following:

1- Pool libraries in pM ratios based on required output, they do not have to be equimolar
2- Libraries pooled in one lane should have compatible (color balanced) indices and good distance in sequence to avoid miss-assignments of reads
3- Libraries with larger fragment sizes will have less read output if pooled with shorter libraries
4- Required sequencing primers for R2 and index reads should be the same for all libraries in one flow cell. Read 1 sequencing primers can be different for different lanes.

Number 4 is not strictly true. You can mix multiple primers in the appropriate R2 & Index primer tubes for the HiSeq. In fact the reagents as delivered from Illumina already contain a mixture of primers for TruSeq DNA/RNA, TruSeq small RNA and Nextera libraries. It doesn't matter that the additional primers will be injected into lanes where they are not used, so long as the primers are different enough from each other to prevent off target binding it's fine.

[nucacidhunter]

Number 4 is not strictly true. You can mix multiple primers in the appropriate R2 & Index primer tubes for the HiSeq. In fact the reagents as delivered from Illumina already contain a mixture of primers for TruSeq DNA/RNA, TruSeq small RNA and Nextera libraries. It doesn't matter that the additional primers will be injected into lanes where they are not used, so long as the primers are different enough from each other to prevent off target binding it's fine.

You are right it is technically possible, but I think a sequencing centre (focus of the tread) would not risk doing this if they have customer prepared libraries requiring custom primers for reads other than R1 for some libraries because of possible interaction among primers which could result in failure or suboptimal sequencing output. It would be OK if the whole flow cell run is for the customer and they accept the risk. Illumina primer mixes have been designed and validated to be compatible.