Dear respected members,

I hope you can shed some light to problems i am currently having. A link to journals related to problem i am having is very much appreciated. I am sorry if my questions are so many and so long. 

I am trying to do smallRNA sequencing from exosomal RNA. The samples are plasma and tissue of cancer patients. We use Life Technologies' Total exosome isolation kit and Total RNA isolation kit. In the beginning, we use nanodrop to measure total RNA post isolation, then move to  qubit fluorometer. Both tools show very very low results. Then the staff here try to measure one miRNA (miR-486-5p) using Taqman miRNA assay,  that was expressed in most samples then use that as proxy of total RNA quantity. 

After total RNA isolation, we proceed with Truseq small RNA library kit (by illumina) to make NGS library. post adapter ligation steps, we quantify the smallRNA using bioanalyzer. the results for plasma always lower than tissue. Then we did gel electrophoresis to purify RNA, then cut the 140-160 bp area of the gel (smallRNA area) and proceed to next steps up to sequencing in MiSeq. 

We have done 7 NGS using MiSeq, each with different number of samples. sometime we processed 8 samples, sometime12 samples, other time 27 samples. 

We then get the total reads results from MiSeq reporter. Mostly, plasma samples showed lower total reads than tissue samples. 

on the last run, several of plasma samples show very very low total reads. usually our plasma samples give more than 300.000 reads. but several samples showed less than 50.000 reads. other extreme results for several samples were less than 100 reads produced. to add the trouble, miRNA reads were less than 50 for several samples, while usually we got 10.000 reads up to 300.000 miRNA reads. 

For samples that produced less than 50 miRNA reads, the confusion occured because the result of Taqman miRNA assay was higher than other samples which has lower Taqman miRNA assay but produced higher total reads and total miRNA reads. 

Examples : 

Sample A : Taqman miRNA assay result (post isolation of exosomal RNA): 0.08 ng/microliter. Post adapter-ligation bioanalyzer result : 0 pmol/l

                     Total MiSeq reads : 166.569. Total miRNA reads : 147 

Sample B : Taqman miRNA assay result (post isolation of exosomal RNA) : 0 ng/microliter

                     Total MiSeq reads : 31.859. Total miRNA reads : 45. Post adapter-ligation bioanalyzer result : 0 pmol/l

Sample C : Taqman miRNA assay result : 1.17 ng/microliter. (post isolation of exosomal RNA)

                      Total MiSeq reads : 32.773. Total miRNA reads : 1797. Post adapter-ligation bioanalyzer result : 0 pmol/l

Questions : 

1. do you ever encounter the same problems of different total reads results with different RNA quantity? and is there linear relationship between total RNA quantity and total reads result? do high RNA quantity always produce high reads?

2. Considering there isn't any certain pattern or relationship between TaqMan miRNa result and total MiSeq reads, is it okay or right to use Taqman miRNA assay as proxy for total RNA result? considering the previous measurement tools always show very low result. 

3. Is there any convention on how many numbers of samples should be run on one NGS run?? My supervisor worried with running 27 samples of plasma would make potential average reads in each sample will be lower than if little samples used. 

4. Is it okay to sequence plasma and tissue samples in one NGS run? Illumina Staff said it is fine, but my supervisor thought because tissue samples always produce higher reads result, then it is assumed that tissue samples will dominate the flow cell if it was ran together with plasma samples which always produce lower results (assumed the plasma exosomal-rna content was lower than tissue)

3. the other samples processed on the same NGS run showed 1000x higher total reads. My supervisor thought that this was because the problem with sampling preparation and related to patients background, considering on the same day of NGS run, tissue samples and plasma samples from same several individuals (lets say patient F, J, K) produced 1000x or more higher reads than tissue and plasma samples of other several individuals (lets say patient G,L,M) which shows higher tissue reads but very low plasma reads, protocols are the same. i can not find any correlation with patients or disease background because in other day run, different cancer staging give different reads result. other than sampling preparation, is there any other issue that might cause low NGS reads. 

Once again, thank you very much for those who read this question, and could give insights or journal-link to questions i am having. 

Regards.