4.2 Working with SAM/BAM files

Question 1

What is the first part of any SAM file?

Answer 1

Header: contains info related to reference sequence dictionary and program used to make the BAM file

Question 2

What is in each of the 11 columns in SAM/BAM?

Answer 2

1. QNAME: Query template Name
2. FLAG: bitwise Flag
3. RNAME: reference sequence name
4. POS: 1-based leftmost mapping position
5. MAPQ = mapping quality
6. CIGAR = Cigar string
7. RNEXT = Ref. name of the mate/next read
8. PNEXT = position of mate/next read
9. TLEN = observed template length
10. SEQ segment sequence
11. QUAL = ASCII pf phred based quality +33

Question 3

What is a bit flag?

Answer 3

An encoding of alignment info; can be used to filter reads downstream

Question 4

What is secondary alignment?

Answer 4

the same part of the sequence aligns to multiple locations

Question 5

What is supplementary alignment?

Answer 5

where (mostly) non-overlapping parts of a sequence align to multiple locations

Question 6

What is a cigar string?

Answer 6

• compresses info about alignments such as matches, insertions, deletions in the order the occur
• encodes alignment segments

Question 7

What does this cigar string mean?

3M1I3MD5M

Answer 7

3 match, 1 insertion, 3 match, 1 deletion, 5 match

Question 8

What to +/- signs in the observed template length indicate?

Answer 8

+: forward read (left most read)

- :reverse read (right most read)

Question 9

If all segments are mapped to the same reference, what does the unsigned observed template length equal to

Answer 9

the number of bases from the left most mapped base to the right most mapped based of the read pairs

Question 10

what does an observed template length of 0 mean?

Answer 10

the template is a single segment of the information in N/A

Question 11

What is Samtools?

Answer 11

A set of utilities that manipulates alignments in BAM format
Used for
-sorting, merging, indexing, read retrieval

checks the working dir for index file and download index if absent

Question 12

What 3 steps are needed to work effectively with SAM files?

Answer 12

1. convert to binary format –> faster for computer to access
2. sort by reference position
3. generate an index to speed look up process

Question 13

What does the samtools sort cmd do?

Answer 13

• sorts alignments by leftmost coordinate

- may create tmp files when whole genome alignment can’t be fitted to memory

Question 14

What command produced a .bai file

Answer 14

samtools index

Question 15

What 3 files are needed to view the alignments in IGV?

Answer 15

1. reference
2. BAM files w alignments
3. index file (bai)