Hello, I am new to the RNAseq. I sequenced 20 libraries from Eucalyptus. They are all different tissues: early flower, late flower pollinated, late flower unpollinated, early seed capsule, late seed capsule, mature pollen, and mature leaf. I have three biological replicates for all the tissues except pollen; I only have two for pollen.
I started my analysis using Hisat2, stringtie, and ballgown. But, I have not figured out how to do multiple pairwise comparisons in ballgown yet. Any suggestions?
Now, I have moved to using DESeq2 instead of ballgown. However, I am not sure how to design the formula for DESeq2 since I only have two replicates for pollen. The design: tissue*tree gets an error because "the model matrix is not full rank". I read on a blog post to combine tree and tissue into one. However, that makes it seems like there are no replicates. I am not sure how to go forward without replicates.
Also, I am concerned that my counts are not normalized well-enough by calculating FPKM values. My reads are single-ended. Is it possible to generate RPKM values using ballgown or DESeq2?
Last, is STAR considered a better aligner than Hisat2?