Hi I am new to Biostars but I have run into a problem where I do not have the expertise to solve.
I performed RNA capture-sequencing on a bunch of cancer samples which means only a select panel of genes are sequenced. With this data I would have liked to apply common across sample normalisation methods such as RLE (Deseq) and TMM however being that this dataset is only 250 genes which were chosen due to their involvement in cancer I worry that they may not follow the assumptions that underly those methods (the main one being that the majority of genes in a sample are not differentially expressed).
I do have ERCCs spiked into the samples. While the intention was to have ERCCs spiked in at relatively similar proportions, some samples ended up with a larger proportion of reads mapping to the ERCCs. Can I still use these ERCCs for normalisation through RUV? Are there any other methods of normalisation? Without a control or any biological replicates how can I check if the normalisation has worked?