Click here to DOWNLOAD SPCR 3.0!

Cases ever used to test and SPCR program

•(1)  Predict 16S rRNA gene from bacteria genomes

Primer sequences:

Primer1: GAGAGTTTGATCCTGGCTCAG

Primer2: CTACGGCTACCTTGTTACGA

Parameters:

I up =0.85 ， I dn =0.85 ， P a =0.85 ， L max =3200bp ， L min =200bp

Templates:

Select completed genome sequences of 59 eubacteria as templates, they are included in table as follow:

Results:

The number, location and direction of 16S rRNAs in 52 prediction results is completely same as data in GenBank, and some variances found in other 7 bacteria. The variances and reasons listed as follow.

Verification:

We verified the 7 different results through sequence alignment and BLAST, and found the reasons that caused those variances.

1. BBUR Borrelia burgdorferi : Through alignment and BLAST analysis, the direction of the only one 16S rRNA gene was mislabeled in GenBank data. This 16S rRNA gene should be located in minus strand of genome, not in plus strand.
2. Clostridium tetani E88 : Through alignment analysis, one of 16S rRNA sequence was mislabeled as 5S rRNA in GenBank data.
3. Lactococcus lactis : The results are same as GenBank when change parameters to: I up =0.9; I dn =0.9; I ave =0.9
4. Salmonella enterica subsp. Typhi : Same as GenBank when change parameters to: I up =0.9; I dn =0.9; I ave =0.9
5. Ureaplasma urealyticum : Same as GenBank when change parameters to: I up =0.86; I dn =0.86; I ave =0.86
6. Vibrio cholerae : Two minus strand 16S rRNAs were mislabeled as plus strand in GenBank data.
7. Yersinia pestis strain CO92 : When reduce thresholds to 0.78, we predicted 6 products, and their locations in genome is consensus with GenBank data, but the downstream primer of the last prediction product can not dock to template correctly, the coefficient is only 0.8, and the product length is 2603bp. This is only one uncertain product that can not be corrected in this case.

• (2) Predict SOX gene family from homo sapiens genome

Primer sequences:

Primer1: CCMATGAAYGCSTTYATSGTSTGG

Primer2: GGYYKRTAYTTRTARTYSGG

Parameters:

I up =0.9, I dn =0.9, P a =0.9, L max =800, L min =80

Templates:

Homo sapiens whole genome (build 25)

Results:

Verification:

Through sequence alignment with SOX gene family and BLAST analysis, each of SPCR products can found their counterpart in SOX gene family. But the number of SOX gene in human genome seems more than 10, so we decreased the value of parameters group to I up =0.85, I dn =0.85, P a =0.85, L max =800, L min =80, but we get only 2 more SOX homologous gene and other 29 unrelated products.

•  (3) Predict ARR5, ARR7 and GEN12, GEN13 gene family (VPCR)

We repeated the experiments used in VPCR paper with our SPCR program, the results are much better than VPCR.

Primer sequences:

ARR5:

ARR5a = GTTGATTCTCTCTATCTCTCTCACG

ARR5b = CACACCACCATTTTACATATCTC

ARR7:

ARR7a = GTTGGTGAGGTCATGAGGATGGAGATTC

ARR7b = GTTTTGCTAAGGTCTTGGCCTCTATACAT

GEN12:

GEN1 = CATGTTCTTGCYGTYGATGAYAGT

GEN2 = CCAGTCATKCCAGGCATWSAG

GEN13:

GEN1 = CATGTTCTTGCYGTYGATGAYAGT

GEN3 = ATAARAAATCYTCAGCWCCTTC

Parameters:

ARR5 and ARR7: I up =0.90 ， I dn =0.90 ， P a =0.90 ， L max =3200bp ， L min =200bp

GEN12 and GEN13: I up =0.85 ， I dn =0.85 ， P a =0.85 ， L max =3200bp ， L min =200bp

Templates:

Arabidopsis thaliana genome sequences

Results:

Prediction of ARR5 and ARR7

Prediction of GEN12

Prediction of GEN13

Verification:

SPCR predicted ARR5 and ARR7 gene exactly from Arabidopsis thaliana genome sequences. In GEN12 and GEN13 cases, SPCR predicted 6 products with GEN12 and 7 products with GEN13. Through BLAST analysis, we can prove all of predicted GEN12 and GEN13 sequences are ARR family gene. This result is much better than VPCR, we almost can not find any significant relationship between VPCR prediction results and real world PCR control, and at the same time, the results of SPCR can match with real world PCR almost perfectly.

•  (4) Predict products of ERIC-PCR from E. coli genome

Primer sequences:

Primer 1(ERIC2): AAGTAAGTGACTGGGGTGAGCG

Primer 2(ERIC1R): ATGTAAGCTCCTGGGGATTCAC

Parameters:

I up =0.80 ， I dn =0.80 ， P a =0.70 ， L max =3200bp ， L min =200bp

Templates:

E. coli MG1655 genome sequence

Results:

Prediction of E. coli ERIC-PCR

Verification:

ERIC-PCR is a kind of random amplified PCR; the process in molecular level is very complex so it's very hard to find a proper mathematical model to describe this process, and very difficult to simulate this process with computational methods. But in spite of this, the counterparts of most of significant bands of real ERIC-PCR pattern can be found in SPCR prediction pattern.

(5) Predict PCR product of Coronavirus genomes

Primer sequences:

Primer pair1 ：

upstream ： IN-2(+) ： 5'-GGGTTGGGACTATCCTAAGTGTGA-3'

downstream ： IN-4(-) ： 5'-TAACACACAACNCCATCATCA-3'

Primer pair 2 ：

upstream ： 2Bp: 5'- ACTCARWTRAATYTNAAATAYGC-3'

downstream ： 4Bm: 5'- TCACAYTTWGGATARTCCCA-3'

Parameters:

I up =0. 90 ， I dn =0. 90 ， P a =0. 90 ， L max =3200bp ， L min =200bp

Templates:

7 complete coronavirus genomes

1. Porcine epidemic diarrhea virus ( 28,033 bp)

2. Human coronavirus 229E ( 27,317 bp)

3. Transmissible gastroenteritis virus ( 28,586 bp)

4. Murine hepatitis virus ( 31,100 bp)

5. Bovine coronavirus ( 31,276 bp)

6. Avian infectious bronchitis virus ( 27,608 bp)

7. SARS coronavirus ( 29,751 bp)

Results :

The result of PCR production of 7 complete coronavirus genomes using SPCR software prediction.

The PCR product length using primer pair1 is 453bp ； The PCR product length using primer pair2 is 251bp

Templates:

14 other coronavirus species containing polymerase gene

1. Murine-Cov open reading frame 1a (gene 1), complete cds and open reading frame 1b (gene 1), 3' end.

2. Human coronavirus 229E mRNA for RNA polymerase and proteases.

3. Transmissible gastroenteritis virus (Purdue-115) mRNA for polymerase locus.

4. Mouse hepatitis virus RNA for viral polymerase open reading frame 1b.

5. Bovine coronavirus RNA-directed RNA polymerase (pol) gene

6. Avian infectious bronchitis virus mRNA for chimeric gene.

7. Human coronavirus (strain OC43) RNA-directed RNA polymerase (pol) gene.

8. Turkey coronavirus RNA-directed RNA polymerase (pol) gene.

9. Rat sialodacryoadenitis coronavirus RNA-directed RNA polymerase (pol) gene.

10. Porcine hemagglutinating encephalomyelitis virus RNA-directed RNA polymerase (pol) gene.

11. Feline infectious peritonitis virus RNA-directed RNA polymerase (pol) gene.

12. Porcine transmissible gastroenteritis virus RNA-directed RNA polymerase (pol) gene.

13. Infectious bronchitis virus RNA (defective RNA CD-61).

14. Canine coronavirus RNA-directed RNA polymerase (pol) gene, partial cds.

Results :

The result of 14 other coronavirus species containing polymerase gene using SPCR software prediction. The product length using primer pair1 is 453bp ； The product length using primer pair2 is 251bp

Verification:

The following articles prove our results ( Ksiazek, Erdman et al. 2003 ; Stephensen, Casebolt et al. 1999 ). Some of the SPCR prediction results have also been verified by our lab. (We have amplified IBV and TGEV in our web lab experiment. The wet lab result proved that the PCR production of IBV and TGEV conform to our SPCR prediction)