I work with fastq files containing NGS reads for some human DNA regions. The reference genome is hg19. I had two fastq files (pair-ended). I generated alignment BAM files. I used "bwa" and samtools to find a possible target gene region (chr7:55,242,376-55,242,574,). This corresponds to a region of the EGFR gene.
Here is a screenshot of the gene region
And here is a screenshot of the primer region
I also have a list of primers and reverse primers:
FORWARD 1. TTGCCAGTTAACGTCTTCCTTCTCTCTCTG 2. CCCTTGTCTCTGTGTTCTTGTCCCCCCCA 3. TGATCTGTCCCTCACAGCAGGGTCTTCTCT 4. CACACTGACGTGCCTCTCCCTCCCTCCA REVERSE 1. GAGAAAAGGTGGGCCTGAGGTTCAGAGCCA 2. CCCCACCAGACCATGAGAGGCCCTGCGGCC 3. TGACCTAAAGCCACCTCCTTA 4. CCGTATCTCCCTTCCCTGATTA
And I have some adaptors
ADAPTORS 1 AAGACTCGGCAGCATCTCCA ADAPTORS 2 GCGATCGTCACTGTTCTCCA
I have to answer to the following questions:
1) What is the constitution of each read (adaptor+primer+amplified region)? The forward primer is obvious and corresponds to the first primer of the list
So why do we have the three other forward primers? I don't understand. And what about the correct reverse primer?
It seems to be the complementary sequence of the end region
Finally which adaptor is used?
Is [ADAPTOR1 - PRIMER1 - AMPLIFIED REGION] the correct answer? Are there other possibilities?
2) % of reads that map the human genome? what about the unmapped?
Can you please give me some clues about how to answer this question.
I just need some clues I want to do it by myself.
Thank you very much for your help.