how to use bwa? and how to set paremeters for 75bp reads

[Tlexander]

Hello everybody.
i'm a new user of bwa to align solexa reads to the reference genome hg19.i have finished bwa index for the hg19 genome,but not very clear how to use bwa aln command,especially all the paremeters in bwa aln:
i want to know the following conception such as:

1. what's seed length? whether it is equal to reads length,my reads length is 75bp

2. how to set number of threads? How much is best?

3. how to set -q INT quality threshold for read trimming down to 35bp [0]
why 35bp?
how to set this paremeter for my 75bp reads.

4. which options are essential just like the following paremeters?

thank you all very much. yours sincerely Alex. your helps are highly appreciated.

my email is [email protected]




bwa aln ********************
Options: -n NUM max #diff (int) or missing prob under 0.02 err rate (float) [0.04]
-o INT maximum number or fraction of gap opens [1]
-e INT maximum number of gap extensions, -1 for disabling long gaps [-1]
-i INT do not put an indel within INT bp towards the ends [5]
-d INT maximum occurrences for extending a long deletion [10]
-l INT seed length [32]
-k INT maximum differences in the seed [2]
-m INT maximum entries in the queue [2000000]
-t INT number of threads [1]
-M INT mismatch penalty [3]
-O INT gap open penalty [11]
-E INT gap extension penalty [4]
-R INT stop searching when there are >INT equally best hits [30]
-q INT quality threshold for read trimming down to 35bp [0]
-c input sequences are in the color space
-L log-scaled gap penalty for long deletions
-N non-iterative mode: search for all n-difference hits (slooow)

[krobison]

Try taking a random slice of your data & play with parameters. All of this is tuning -- the defaults will give you reasonable results.

The number of threads is almost easy: no more than the number of processors (cores) you actually have. BFAST suggests using a power of 2 but bwa doesn't mention this; I don't know if it matters in that case. More threads is faster, by a not quite linear amount.

[Tlexander]

thank you very much.
i find another question in the process.
Can you tell me the function of the bwa index? which result or file generated by bwa index were used in the following process? i find nothing of the bwa index result was used in the following bwa aln and bwa samse.et.al.
i want to know why?

[wisosonic]

seed is the first INT nucleotides in your read that usually have high quality .. so you can play with the length of the seed and the number of #diff in it.

the index is a way to make alignement very fast by creating 8 files from your reference sequence. so when doing the alignement the program will have just the needed info from the index without returning to the reference sequence that might be very long.