Hi, I am trying to do RNA-seq de novo on a polyploid (hexaploid) genome using a combination of 454 and illumina 100bp paired ended reads. The three copies of each gene are highly similar to one another. I am having trouble in separating each one of the three copies into three different fully-length assemblies. Most of the times I just get a fragment of each of the three copies. I am guessing that when Mira finds a difference between highly similar transcripts, it just can't assess if there is a polymorphism between 2 of the copies or a sequencing error. In any case, Mira seems to end the assembly way before reaching the end of the transcript. I have prepared and run 2 different Manifests. I am getting better results with Manifest.conf (less number of contigs but longer) than with Manifest2.conf (higher number but shorter contigs), so I am supposing that I could eventually separate the 3 copies of each gene by fine-adjusting the parameters. Any suggestion would be greatly appreciated, Thanks so much! Juan This electronic message contains information generated by the USDA solely for the intended recipients. Any unauthorized interception of this message or the use or disclosure of the information it contains may violate the law and subject the violator to civil or criminal penalties. If you believe you have received this message in error, please notify the sender and delete the email immediately.
Localtime: Tue Jan 21 03:11:57 2014
Assembly information:
=====================
Localtime: Tue Jan 21 03:11:57 2014
MIRA version: 4.0rc4
Num. reads assembled: 25787099
Num. singlets: 0
Coverage assessment (calculated from contigs >= 1000 with coverage >= 12):
=========================================================
Avg. total coverage: 30.84
Avg. coverage per sequencing technology
Sanger: 0.00
454: 0.78
IonTor: 0.00
PcBioHQ: 0.00
PcBioLQ: 0.00
Text: 0.00
Solexa: 28.34
Solid: 0.00
Large contigs (makes less sense for EST assemblies):
====================================================
With Contig size >= 500
AND (Total avg. Cov >= 10
OR Cov(san) >= 0
OR Cov(454) >= 0
OR Cov(ion) >= 0
OR Cov(pbh) >= 0
OR Cov(pbl) >= 0
OR Cov(txt) >= 0
OR Cov(sxa) >= 9
OR Cov(sid) >= 0
)
Length assessment:
------------------
Number of contigs: 67143
Total consensus: 71552356
Largest contig: 9612
N50 contig size: 1161
N90 contig size: 616
N95 contig size: 559
Coverage assessment:
--------------------
Max coverage (total): 184917
Max coverage per sequencing technology
Sanger: 0
454: 180830
IonTor: 0
PcBioHQ: 0
PcBioLQ: 0
Text: 0
Solexa: 61823
Solid: 0
Quality assessment:
-------------------
Average consensus quality: 81
Consensus bases with IUPAC: 49550 (you might want to
check these)
Strong unresolved repeat positions (SRMc): 0 (excellent)
Weak unresolved repeat positions (WRMc): 322 (you might want to
check these)
Sequencing Type Mismatch Unsolved (STMU): 0 (excellent)
Contigs having only reads wo qual: 0 (excellent)
Contigs with reads wo qual values: 0 (excellent)
All contigs:
============
Length assessment:
------------------
Number of contigs: 480338
Total consensus: 175708905
Largest contig: 9612
N50 contig size: 545
N90 contig size: 165
N95 contig size: 133
Coverage assessment:
--------------------
Max coverage (total): 184917
Max coverage per sequencing technology
Sanger: 0
454: 180830
IonTor: 0
PcBioHQ: 0
PcBioLQ: 0
Text: 0
Solexa: 61823
Solid: 0
Quality assessment:
-------------------
Average consensus quality: 66
Consensus bases with IUPAC: 138517 (you might want to
check these)
Strong unresolved repeat positions (SRMc): 0 (excellent)
Weak unresolved repeat positions (WRMc): 380 (you might want to
check these)
Sequencing Type Mismatch Unsolved (STMU): 0 (excellent)
Contigs having only reads wo qual: 0 (excellent)
Contigs with reads wo qual values: 0 (excellent)
Localtime: Fri Feb 21 03:18:07 2014
Assembly information:
=====================
Localtime: Fri Feb 21 03:18:07 2014
MIRA version: 4.0
Num. reads assembled: 12655197
Num. singlets: 0
Coverage assessment (calculated from contigs >= 1000 with coverage >= 12):
=========================================================
Avg. total coverage: 41.27
Avg. coverage per sequencing technology
Sanger: 0.00
454: 2.49
IonTor: 0.00
PcBioHQ: 0.00
PcBioLQ: 0.00
Text: 0.00
Solexa: 36.21
Solid: 0.00
Large contigs (makes less sense for EST assemblies):
====================================================
With Contig size >= 200
AND (Total avg. Cov >= 21
OR Cov(san) >= 0
OR Cov(454) >= 1
OR Cov(ion) >= 0
OR Cov(pbh) >= 0
OR Cov(pbl) >= 0
OR Cov(txt) >= 0
OR Cov(sxa) >= 18
OR Cov(sid) >= 0
)
Length assessment:
------------------
Number of contigs: 67511
Total consensus: 39791618
Largest contig: 5919
N50 contig size: 609
N90 contig size: 399
N95 contig size: 314
Coverage assessment:
--------------------
Max coverage (total): 3910
Max coverage per sequencing technology
Sanger: 0
454: 3726
IonTor: 0
PcBioHQ: 0
PcBioLQ: 0
Text: 0
Solexa: 2921
Solid: 0
Quality assessment:
-------------------
Average consensus quality: 55
Consensus bases with IUPAC: 9244 (you might want to
check these)
Strong unresolved repeat positions (SRMc): 0 (excellent)
Weak unresolved repeat positions (WRMc): 5 (you might want to
check these)
Sequencing Type Mismatch Unsolved (STMU): 0 (excellent)
Contigs having only reads wo qual: 0 (excellent)
Contigs with reads wo qual values: 0 (excellent)
All contigs:
============
Length assessment:
------------------
Number of contigs: 826277
Total consensus: 194908914
Largest contig: 5919
N50 contig size: 234
N90 contig size: 150
N95 contig size: 135
Coverage assessment:
--------------------
Max coverage (total): 3910
Max coverage per sequencing technology
Sanger: 0
454: 3726
IonTor: 0
PcBioHQ: 0
PcBioLQ: 0
Text: 0
Solexa: 2921
Solid: 0
Quality assessment:
-------------------
Average consensus quality: 58
Consensus bases with IUPAC: 13244 (you might want to
check these)
Strong unresolved repeat positions (SRMc): 0 (excellent)
Weak unresolved repeat positions (WRMc): 5 (you might want to
check these)
Sequencing Type Mismatch Unsolved (STMU): 0 (excellent)
Contigs having only reads wo qual: 0 (excellent)
Contigs with reads wo qual values: 0 (excellent)
Attachment:
Manifest2.conf
Description: Manifest2.conf
Attachment:
Manifest.conf
Description: Manifest.conf
