De novo assembly for virus genome with Velvet.

Question

[Peter]

Dear expert,

I want to assembly a virus genome using velvet. The virus genome is about 3KB length. In the follow command, ref1.fa is the reference genome. But it seems that I can not success. I cannot get back ref1.fa from the simulate reads.

~/bin/bioinfomatics/wgsim-master/wgsim -N 500000 -1 100 -2 100 -h ~/projects/virus/analysis/ref1.fa r1.fq r2.fq
~/bin/bioinfomatics/velvet_1.2.10/contrib/shuffleSequences_fasta/shuffleSequences_fasta.pl r1.fq r2.fq output.fq

~/bin/bioinfomatics/velvet_1.2.10/contrib/VelvetOptimiser-2.2.4/VelvetOptimiser.pl\
    -s 27 -e 31 -f '-longPaired -fastq output.fq' -t 4 --optFuncKmer 'n50'

Dec  1 17:18:33
Will run velvet optimiser with the following paramters:
    Velveth parameter string:
        -shortPaired -fastq output.fq
    Velveth start hash values:    27
    Velveth end hash value:        31
    Velveth hash step value:    2
    Velvetg minimum coverage cutoff to use:    0

    Read tracking for final assembly off.
Dec  1 17:18:33

    Beginning velveth runs.
********************************************************
Assembly id: 1
Velveth timestamp: Dec  1 2014 17:18:57
Velveth version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_27 27 -shortPaired -fastq output.fq
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_27
Velvet hash value: 27
Roadmap file size: 110519999
**********************************************************
********************************************************
Assembly id: 2
Velveth timestamp: Dec  1 2014 17:18:59
Velveth version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_29 29 -shortPaired -fastq output.fq
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_29
Velvet hash value: 29
Roadmap file size: 107811920
**********************************************************
********************************************************
Assembly id: 3
Velveth timestamp: Dec  1 2014 17:19:00
Velveth version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_31 31 -shortPaired -fastq output.fq
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_31
Velvet hash value: 31
Roadmap file size: 103399704
**********************************************************
Dec  1 17:19:00

    Beginning vanilla velvetg runs.
********************************************************
Assembly id: 1
Assembly score: 53
Velveth timestamp: Dec  1 2014 17:18:57
Velvetg timestamp: Dec  1 2014 17:21:52
Velveth version: 1.2.10
Velvetg version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_27 27 -shortPaired -fastq output.fq
Velvetg parameter string: auto_data_27  -clean yes
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_27
Velvet hash value: 27
Roadmap file size: 110519999
Total number of contigs: 1062
n50: 53
length of longest contig: 95
Total bases in contigs: 58657
Number of contigs > 1k: 0
Total bases in contigs > 1k: 0
**********************************************************
********************************************************
Assembly id: 2
Assembly score: 57
Velveth timestamp: Dec  1 2014 17:18:59
Velvetg timestamp: Dec  1 2014 17:22:06
Velveth version: 1.2.10
Velvetg version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_29 29 -shortPaired -fastq output.fq
Velvetg parameter string: auto_data_29  -clean yes
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_29
Velvet hash value: 29
Roadmap file size: 107811920
Total number of contigs: 424
n50: 57
length of longest contig: 95
Total bases in contigs: 25334
Number of contigs > 1k: 0
Total bases in contigs > 1k: 0
**********************************************************
********************************************************
Assembly id: 3
Assembly score: 61
Velveth timestamp: Dec  1 2014 17:19:00
Velvetg timestamp: Dec  1 2014 17:22:07
Velveth version: 1.2.10
Velvetg version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_31 31 -shortPaired -fastq output.fq
Velvetg parameter string: auto_data_31  -clean yes
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_31
Velvet hash value: 31
Roadmap file size: 103399704
Total number of contigs: 1917
n50: 61
length of longest contig: 99
Total bases in contigs: 121773
Number of contigs > 1k: 0
Total bases in contigs > 1k: 0
**********************************************************
Dec  1 17:22:07 Best assembly by assembly score - assembly id: 3
Dec  1 17:22:07 Optimisation routine chosen for best assembly: shortPaired
Dec  1 17:22:07 Looking for the expected coverage
Dec  1 17:22:09        Expected coverage set to 0
********************************************************
Assembly id: 3
Assembly score: 61
Velveth timestamp: Dec  1 2014 17:19:00
Velvetg timestamp: Dec  1 2014 17:22:07
Velveth version: 1.2.10
Velvetg version: 1.2.10
Readfile(s): -shortPaired -fastq output.fq
Velveth parameter string: auto_data_31 31 -shortPaired -fastq output.fq
Velvetg parameter string: auto_data_31  -clean yes -exp_cov 0
Assembly directory: /Users/jhuang/projects/hbv/analysis/auto_data_31
Velvet hash value: 31
Roadmap file size: 103399704
Total number of contigs: 1917
n50: 61
length of longest contig: 99
Total bases in contigs: 121773
Number of contigs > 1k: 0
Total bases in contigs > 1k: 0
Paired Library insert stats:
**********************************************************
Dec  1 17:22:09 Setting the short insert length
Dec  1 17:22:09 Setting assembly short insert length(s) to auto
Dec  1 17:22:09 Beginning coverage cutoff optimisation
Minimum specified coverage cutoff is higher than the expected coverage. Please choose a minimum coverage cutoff smaller than 0 and re-run.

[Peter]

Here is my reference.

>2547-16_ASC_B
CTCCACCACTTTCCACCAAACTCTTCAAGATCCCAGAGTCAGGGCCCTGTACTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTGAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTACCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACAACCAGCACCGGACCATGCAAGACCTGCACAACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGTTATATGGATGATGTGGTTTTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACCCATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAACAAAAAGATGGGGATATTCCCTTAACTTCATGGGATATGTAATTGGGAGTTGGGGCACATTGCCACAGGAACATATTGTACAAAAAATCAAAATGTGTTTTAGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTCTTTTGGGGTTTGCCGCACCTTTCACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGCATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTCAACAGTATTTGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGGGCAAAACTCATCGGGACTGACAATTCTGTCGTGCTCTCCCGCAAGTATACATCATTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGCCTATTGTACCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGCCCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACAGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTAATGAGTGGGAGGAGTTGGGGGAGGAGGTGAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTGTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCGACACCGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCAACCTGGGTGGGAAGTAATTTGGAAGATCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAACGTTAACATGGGCCTAAAAATCAGACAACTATTGTGGTTTCATATTTCCTGTCTTACTTTTGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCTGCATATAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAGTATTCCTTGGACACATAAGGTGGGAAACTTTACGGGGCTTTATTCTTCTACGGTACCCTGCTTTAATCCTAAATGGCAAACTCCTTCTTTTCCCGACATTCATTTGCAGGAGGACATTGTTGATAGATGTAAGCAATTTGTGGGGCCCCTTACAGTAAATGAAAACAGGAGACTAAAATTAATTATGCCTGCTAGGTTTTATCCCAATGTTACTAAATATTTGCCCTTAGATAAAGGGATCAAACCGTATTATCCAGAGTATGTAGTTAATCATTACTTCCAGACGCGACATTATTTACACACTCTTTGGAAGGCGGGGATCTTATATAAAAGAGAGTCCACACGTAGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGATCTACAGCATGGGAGGTTGGTCTTCCAAACCTCGAAAAGGCATGGGGACAAATCTTTCTGTCCCCAATCCCCTGGGATTCTTCCCCGATCATCAGTTGGACCCTGCATTCAAAGCCAACTCAGAAAATCCAGATTGGGACCTCAACCCGCACAAGGACACCTGGCCGGACGCCAACAAGGTGGGAGTGGGAGCATTCGGGCCAGGGTTCACCCCTCCCCATGGGGGACTGTTGGGGTGGAGCCCTCAGGCTCAGGGCCTACTCGCAACTGTGCCAGCAGCTCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACACTCATCCTCAGGCCATGCAGTGGAA

Answer 1

[Daniel]

Something appears to be going wrong before that error as none of your contigs are larger than 99 bases. A few points:

• Your input data type is 100bp paired end right? I wouldn't describe that as 'long paired end' in your command. But it seems that velvetoptimiser is running shortpaired anyway, so that may or may not be an issue.
• What does the quality look like on the data? I don't know wgsim, but could the artificial error profiles be screwing it up?

[Peter]

My input data is 100bp paired end.

I simulate the data with wgsim. The data quality is fine on the data.

Answer 2

[rtliu]

The coverage is too deep for velvet to handle, try to reduce the coverge to 50x - 100x. e.g. wgsim -N 1500.

With your current simulated data, use velvet-estimate-exp_cov.pl to evaluate the coverage-cutoff (say 300), add -exp_cov auto -cov_cutoff 300 parameters to velvetg

[Peter]

Why here is not the deeper the better?

The reference genome is about 3000bp. Which is better for 2 * 300bp or 2 * 100bp or 1 * 300bp?

[Yang Li]

Try normalization the reads before assembly. http://ged.msu.edu/papers/2012-diginorm/