LemurLady18

Just a note, bowtie is very specific about its command... before you do tophat command, you need to "repackage" the reference genome file with bowtie. For example, in my lab, we use this command: $bowtie2-build ref_genome.fa ref_genome the fasta file is basically being repackaged so that tophat can use it. But the "package" (ref_genome) has to have the same name as the fasta file (ref_genome.fa), obviously removing the .fa partThen, you can follow this with your tophat command (all in the same shell) Tuxedo Genome Guided Transcriptome Assembly Workshop site gives a good explanation of the workflow for this type of analysis as well: https://github.com/trinityrnaseq/RNASeq_Trinity_Tuxedo_Workshop/wiki/Tuxedo-Genome-Guided-Transcriptome-Assembly-Workshop Alos, you can find a helpful flow diagram in the Cufflinks manual: http://cole-trapnell-lab.github.io/cufflinks/manual/

Just some things to note for velvet: the output file of both velvetg and velveth commands need to be the same keep cov_cutoff low and then play with min_contig_lgth, exp_cov and word-size to get optimum N50 and contig number Just some things to note for NCBI blast: in command line, in order to submit the command script using the contig file produced by velvet, you need to be "positioned" in the velvet output file, otherwise it won't be able to find the contig file (unless you specify the location using a relative root to open the text file produced by blast, use the command "nano file.txt" and it will show up

This is a great explanation! Just to add, here is an example of a trimmomatic command that i used for my paired-end analysis data: $java -jar <path to trimmomatic app> PE -phred33 read1_input.fq read2_input.fq read1_output.fastq read1_output_unpaired.fastq read2_output.fastq read2_output_unpaired.fastq ILLUMINACLIP:<path to adapters>/TruSeq-PE.fa:2:20:3 SLIDINGWINDOW:5:20 HEADCROP:15 MINLEN:40 PE.. if yours in Single-end you use SE ILLUMINACLIP: TruSeq2-PE.fa is the paired-end TruSeq adapters, because my library prep used these 2:20:3 means 2 mismatches are allowed before a match is rejected 2:20:3 means the match between the adapter ligated reads must be above 20 2:20:3 means match between any adapter sequence and read must be above 3 SLINDINGWINDOW: 5:20 means that cutting of the read will happen when the average quality over a window of 5 nucleotides is below Phred 20 - prevents removal of large good quality regions of reads when only one base is badHEADCROP: 15 - trims 15 nucleotides off the 5' end of the readsMINLEN 40 - will discard reads that are less than 40 bases long after trimming has been doneI hope this also helps