Is there a way to check the quality of small RNA library before receiving raw data from small RNA seq?

Question

[jae kyoung]

Hi all, This is my first time learning the world of NGS. I lack a lot of knowledge. Small RNA sequencing is in progress. But we left the blood sample to the company and received raw data. (fastq file) Before receiving raw data, library sequencing caused a result of 'fail' due to mostly a small number of libraries for the entire sample, but we proceeded with sequencing and obtained a fastq file.

I have a question here. So far, I know a little about checking the quality of raw data while learning about NGS. However, I was wondering if there is a way to check the read of small RNA in raw data if the quality results of the library of small RNA appear as a 'fail' before receiving raw data for the first time like this.

To make the results reliable in future analyses, is there a way to check the miRNA read present in raw data (fastq) and check the quality?

Thank you. all. There are so many things I don't know. It may be a basic part, but please comment a lot. It's a valuable answer for me.

Answer 1

[colindaven]

Some core units and companies check the quality of a library (eg bisulfite or chip-seq) before sequencing using a small, cheap flowcell. This gives some information on the quality of the library before the larger, more expensive flowcells are run.

In many cases MiSeq is used first, or flongle for Oxford Nanopore, before Novaseq or Promethion flowcells are used later.

I don't know a lot about library prep for miRNA/sRNA libraries since I haven't had many to deal with so far.

Keep in mind it's important to do many (2-3?) rounds of trimming to make sure the miRNA reads are actually 21 bp (if desired). Check the trimmed data with fastqc and mapping to genome after each iteration before you're happy with results.

[jae kyoung]

Thank you for your answer. IIf so, can I understand that it means that you have to mapping and verify the quality of the miRNA read in the raw data (fastq) file in the end? I have one more question. I understand that 18-30nt is appropriate for read length for small RNA seq. If you want to perform a follow-up analysis by trimming on this basis Is the sequence length in the Basic Statistics of the fastqc result report between 18 and 30? I know the concepts of bp and nt theoretically, but I don't know yet if the length of the result report means nt or bp.

[colindaven]

bp and nt are interchangeable in my experience.

There should be some good tutorials here for smallRNA with more details: https://training.galaxyproject.org/training-material/topics/transcriptomics/

[jae kyoung]

Thank you, the link is helping. Previously, it was recommended to perform 2-3 trimming rounds to verify that the miRNA read is actually the correct miRNA. Does this mean we should proceed with multiple trimming on different criteria and check the data with genome mapping?

[colindaven]

Good. No, multiple rounds, same criteria. Perhaps with trimmomatic. Save each result set in a differerent directory and check them out with fastqc. Perhaps align to the genome to "eyeball" results. It's a bit fiddly but otherwise results can be incorrect (eg 1 bp too many) which someone is bound to point out downstream.

Answer 2

[GenoMax]

On experimental side of things it is best to use a QC kit meant for small RNA libraries like https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-rna-kits-reagents/bioanalyzer-small-rna-analysis-228257 before you ever get to sequencing.

Many small RNA libraries ligate an adapter directly to RNA so there will be specific procedures to detect this adapter and trim it off. Ideally only those reads that contain the small RNA adapter should be considered real RNA.