While in general we desire to have higher coverage, implying higher read lengths in DNA seq to locate to cover the complete region on the DNA or to have overlap between read lengths. (Correct me if i am wrong here, as this is based upon my concept) but i have learnt that RNA seq needs short read lengths, especially true from small micro RNAs.
Can someone confirm that with reasons ?
I would say longer the read length, more the information on hand. To detect the exact expression profile and splicing pattern, longer reads are always preferred. Moreover, for the micro RNA as you mentioned, small reads could be used, but in the case of the bad sequencing (especially in Illumina reverse reads) bases towards the end of sequence, a lot of data is trimmed out and lost. This is majorly the practical problem which is faced by many researches while analyzing the sequencing data.
In case of RNA sequencing, the purpose would decide the right choice of read length and sequencing depth is very important to detect the rare transcripts.
Yes agreed. But letting some of the adopter diners stay will also be a problem& again result in poor quality RIN
In addition to previous answer of mine, the reason why people use short reads for microRNA is that, short reads if 50bp or 75bp gives enough coverage for the miRNA detection. I haven't worked with RNA-seq, therefore I cant claim which length is more suitable practically.
I didn't understand what you mean by adapters and dimers and RIN. Are you talking about quantification or sequencing itself?
Adopters u add while doing the small RNA sequencing
RIN stands for the RNA Integrity Number & is one of the quality metrics and range between 0 to 10 & > 6 is considered satisfactory for further workup.
These added adopters to RNA during template & cDNA library preparation sometimes won't get trimmed during during bioinformatics work up & u end up having very high values like > 20-30% of primer-diners which gives u difficulty in data interpretation.
I didn't ask for the definition of adapters and RIN, and l am well aware of what they are. I meant sequencing and quantification (RIN) are different thing, so if you are talking about sequencing and read length, you should not talk about RIN. In relation to the data analysis, the adapters are generally taken care of during the data processing and any good bioinformatics pipeline could be used for it. If adapters are not removed, it is the problem in script and personal problem. You have to check and modify the scripts according to your project.
Sorry Abhijeet for explaining what u already knew.
I agree the adopter dimer problem occur while processing, & bioinformatics calculated what we did.
Infact I am more interested in quantification which initially showed low miRNA counts.
Regards
what is the minimum read length for Bacterial RNA seq and total number of reads?
I am Very new to RNA Seq work, Can anyone tell me the good length of single read and total number of reads required for good analysis of total transcriptome seq.
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