Hi all, Maybe I'm asking a too basic question, but I really feel confused. I have R1.fastq file and R2.fastq file from the paired-end RNA-seq. As far as I know, the read order in R1 and R2 files should be the same, namely the reads in the same pair should get the same rank in R1 and R2 respectively. However, when I count the initial read numbers in R1 and R2 files, they are different. For example, R1 has 1878678 reads, while R2 has 1800352 reads. This makes me confused becasue if so, does this mean the additional reads in R1 compared to R2 (1878678 - 1800352 = 78326 reads) are unpaired and all the other reads in R1 and R2 are paired and have the same rank? What makes me more confusing is that, after trim R1 and R2 using Trimmomatic (PE mode), the trimmed, and PAIRED R1 and R2 files still have different read numbers. (R1, 1397878, R2, 1402966). So, does this mean the additional reads in R2 this time (1402966 - 1397878 = 5088 reads) are not paired and others are paired with R1? But trimmomatic attributes these reads to the PAIRED result file and actually the unpaired reads have been transferred to the special unpaired fastq result files. This makes me feel confused. Could anyone give some answers? Thank you so much.