Question

Grep all alignments of reads with read id SRR2969247.26811510, and answer the questions below:

What is the position of the forward strand read? How can you tell?

What is the CIGAR of the reverse strand read? What does the CIGAR string tell you?

What is the template length that spans the leftmost position of the first read to the rightmost position of the second read?

What is the mapping quality of both reads?

The forward strand read has a deletion. What is the deletion position, and what is the reference nucleotide in the deletion position?

Answer 1

The question relates to sequencing fragments generated from a template stand 5'CGAGTACG3' in the presence of dideoxynucleotide ddCTP. Fragments of varying lengths are formed, each terminating at a different position where ddCTP is incorporated. These fragments help determine the sequence of the template strand.

Step-by-step explanation:

The student's question pertains to sequencing technology, specifically to the identification and analysis of sequenced reads from a next-generation sequencing dataset, likely originated from the SAM/BAM file format. When performing sequencing, the template strand 5'CGAGTACG3' would be used to generate a series of fragments in the presence of the four deoxynucleotides (dATP, dTTP, dGTP, dCTP) and a dideoxynucleotide (ddCTP). The dideoxynucleotide acts as a chain terminator because it lacks the 3' hydroxyl group necessary for the formation of a phosphodiester bond with the next nucleotide, thus terminating the DNA strand elongation once incorporated.

Fragments that would be generated include:

• A 5'CGA3' fragment (terminated before the first C)
• A 5'CGAGTA3' fragment (terminated before the second C)
• The full 5'CGAGTACG3' strand, as there is no additional C after the second C for ddCTP to terminate

Each fragment has a 3' end, which corresponds to the terminal nucleotide of the sequence that just incorporated the ddCTP, and a 5' end which is the starting point of the template.

The fragments would be used to determine the sequence of the template strand by sizing and identifying the terminal nucleotide of each fragment, giving the complementary sequence to the template strand.