How to check if the total million of reads that I have are the ones that I bought?

Question

[ev97]

I sent some samples to perform RNA-seq and I paid for a total of 100 million of reads (paired end) per sample.

I have done fastqc in those samples and assuming that "Total Sequences" represents the million of reads of each sample, my samples are around 28 to 38 million of reads.

Is it correct to verify that what I received is what I paid if I sum the reads from the two fastq files (_1 and _2)? For example: 28192578 + 28192578 = 56.385.156. --> Therefore, what I received is not what I paid (I have half of what I was expecting).

Is it correct to check this following this way? If not, could you tell me what do you usually do?

Thanks very much in advance

Regards

Answer 1

[GenoMax]

If you were expecting to get 100 M reads per sample then in Illumina speak that would have equated to 50 M clusters leading to 100 M paired-end reads. Did your agreement with the provider specify this specific yield? If it did then you could legitimately ask for a re-run of the sample giving you another set of similar read numbers. Keep in mind that you may simply be oversampling your data when doing this. Complexity of the sample is already pre-decided by the library ( which will not be remade).

If the libraries were of less than optimal quality then it would theoretically be possible to start with ~100M reads and potentially end up with ~58M reads of scanned/trimmed usable data.

[ev97]

Thanks for your quick reply!

What did you mean with clusters? (50 M clusters)

[GenoMax]

Each cluster on a flowcell forms from a unique library fragment during bridge-amplification/clustering. Each cluster is sequenced just from one end (single-end sequencing) or from both ends of the library fragment (paired-end sequencing), which is what you get in the result files (R1/R2). So 50M clusters will lead to 50M single-end or 100M paired end reads.