# --force option in hybrid

y_kim2
Offline
Joined: 03/13/2012

I am using hybrid or hybrid-min with --force option.
What I would like to calculate is the free energy of 3'end-anchored hybridization (3'end 3base exact match).
Suppose the length of the input sequence is n, that can be calculated as
"hybrid --force (n-2),0,3 (sequence_1) (sequence_2)"
or
"hybrid --force 0,n,3 (sequence_2) (sequence_1)"
I was thinking those two give the same results.
However, the both commands produced different results.

So I would like to ask if my understanding is correct.

zukerm
Offline
Joined: 11/12/2010

Your understanding is not correct. The command
`hybrid(-min) --force=(n-2),0,3 (sequence_1) (sequence_2)`
would force the last three bases of the first sequence to hybridize with any three bases of the second sequence. The choice of the bases would be determined by the program to minimize free energy. The notation
`--force i,j,k` forces the base pairs i-j, (i+1)-(j-1)..., (i+k-1)-(j-k+1). Note that in the base pair i-j, i is the base number in the first sequence and j is the base number in the second sequence. If j = 0, then bases i, i+1, ... i+k-1 of the first sequence must form base pairs with some program chosen bases of the second sequence. If i = 0, then bases j, j-1, ... j-k+1 of the second sequence must form base pairs with some program chosen bases of the first sequence.
Suppose that the sequences are
1: 5'-tcgactacggactggcgatctacgcgagctatcgagctacgctACG-3' and
2: 5'-tcgactacggactggcgatctacgcgagctatcgagctacgctCGT-3'
Both have length 46 (n = 46 for both). Then running `hybrid-min` with `--force=44,0,3` gives

```        10        20         30                40
tcgactacggactg    tctac   -    atcg    a--------     ---
gcga     gcg agct    agct         cgctA   CG
TGCt     cgc tcga    tcga         gcggt   gc
-----   a    gcta    gcgcatcta     cag  atcagct
40        30        20        10
```

Running `hybrid-min` with `--force=--force=0,46,3` gives

```        10        20         30        40
tcgactacggactg    tctac   -    atcg    a   tACG
gcga     gcg agct    agct cgc
TGCt     cgc tcga    tcga gcg
-----   a    gcta    -   catctagcggtca...
40        30         20
```

Running `hybrid-min` with `--force=44,46,3` gives

```   20        30        40
...tctacgcgagctatcgagctacgct
ACG
TGC
tcgcatcgagctat...
40        30
```

Do you understand?

I have an important comment to add. By not specifying RNA or DNA, you got the default, which is RNA. The free energies are for RNA using the old version 2.3 free energies at 37 °C. If you are simulating DNA hybridization, then you must use `--NA=DNA`. If you specify no temperature, 37 °C will be used by default. It is almost always better to specify everything on a command line rather than to rely on default values. Run `hybrid(-min) -h` for details and/or look at the man pages.