Question: Salmon choosing library type
0
gravatar for bharata1803
2.6 years ago by
bharata1803490
Japan
bharata1803490 wrote:

Hello,

So, I try to use salmon to quantify the read count from my fastq. I use salmon quant for this method. My question is how can I determine the library type? I know salmon provide automatic lib type detection, but I just want to make sure to choose the right lib type.

From the GEO dataset, the extraction protocol is like this:

RNA was extracted from all samples using the AllPrep DNA/RNA FFPE kit from Qiagen and suspended in nuclease free water (RNA) or AE buffer (DNA). Sample concentrations were measured using NanoDrop 1000 (Thermo Fisher Scientific), and RNA integrity numbers were obtained on the 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer’s protocol Approximately 30 ng of RNA was used based on sample concentrations obtained from the Qubit HS RNA assay (Thermo Fisher Scientific). All samples were reverse-transcribed to generate cDNA libraries using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) adhering to the manufacturer’s protocol for FFPE samples with 16 cycles of target amplification. Library concentrations were determined by qPCR using an absolute quantitation method and the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Template reactions were carried out using the Ion PI Hi-Q OT2 200 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and then loaded onto Ion PI chips v3 using the Ion PI Hi-Q Sequencing 200 Kit based on the manufacturer’s protocol (Thermo Fisher Scientific).

It seems the cDNA libraries is generated using Ion AmpliSeq Transcriptome Human Gene Expression Kit. I tried to read the user guide but can not find how the library is prepared. As for the samples from the GSE102511, most of the salmon detection result is U (unstranded) but some samples are detected as SF (Single-end Forward strand).

They use the same protocol it should be the same for all samples I think.

Anyone familiar with the protocol here can suggest libtype parameter for Salmon? If not I will just go with automatic.

On top of that, how much it will affect the result of read counting if I use wrong lib type for Salmon or other slignment software?

Thank you.

salmon alignment • 1.6k views
ADD COMMENTlink modified 2.6 years ago by igor12k • written 2.6 years ago by bharata1803490

From just reading the kit specifications, I did not see any information on strandedness. From the library information on NCBI, it is single-end sequencing, so I would go for the unstranded setting. Run it and see if the mapping rate is adequate.

ADD REPLYlink modified 2.6 years ago • written 2.6 years ago by ATpoint44k

the automatic result has quite good mapping rate and most of the sample is detected as unstranded. so I think I will ust set it to automatic then.

ADD REPLYlink written 2.6 years ago by bharata1803490
1
gravatar for Antonio R. Franco
2.6 years ago by
Spain. Universidad de Córdoba
Antonio R. Franco4.5k wrote:

Only to complete information for future use

I am not familiar with Salmon, but many different papers relate that both, Kallisto and Salmon provide you with (almost) the same output. Kallisto however, has the options --rf-stranded and --fr-stranded for its use in dUTP or Illumina libraries, respectively

ADD COMMENTlink written 2.6 years ago by Antonio R. Franco4.5k
1
gravatar for igor
2.6 years ago by
igor12k
United States
igor12k wrote:

If you are not sure what the proper strand is, you can try all three options and compare the results. If the library is really stranded, there should be a big difference between the results for the two strands.

You can also check this similar earlier discussion (different tool, but similar issue): featureCounts in predicting strand specificity

ADD COMMENTlink written 2.6 years ago by igor12k
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