Have to agree with this (although in our case SOAPdenovo isn't doing a very good job on our assemblies but it is due to other issues). Depending on coverage and number of reads the 300bp insert library may have generated enough data for a reasonable assembly by itself, and so adding the 600bp insert library won't necessarily net you huge gains. But the gains you have gotten seem pretty good.
Genome Assembly Using 2 Insert Size Libraries
Hi,
I want to assembly ~20Mb fungal genome. We sequenced (illumina, pair-end, 45bp) 2 insert-size libraries: 300bp and 600bp.
I've been using Velvet so far (1.1.04). There is no much improvement between assembly created using only one libraby as compared to assembly using both libraries:
library n50 coverage contigs 300bp 4,314 27.762 4916 both 9,096 79.890 3122
I've tried ABySS as well, but number of contigs >1kb is over 8000. Could you recommend me other assemblers that perform better with multiple insert size libraries?
UPDATE:
I've tried SOAPdenovo - it seems outperform Velvet in case of 2 insert size libraries. Interestingly, Velvet outperforms SOAPdenovo in case of other fungi (very closely related though), but for those we have only one library.
All data generated with k=39 (until stated otherwise). K=39 was found to perform the best accordingly to VelvetOptimiser.
#fn contigs bases GC [%] Ns (%s) N50 N90 SOAP/pe_300only 4060 18918810 61.162 522787 (2%) 12422.45 6149.31 SOAP/pe_600only 4101 18956824 60.876 5499903 (29%) 12141.85 6058.75 velvet/pe_300only 4916 18755580 61.156 492899 (2%) 8488.68 4787.55 velvet/pe_600only 2379 17849192 60.189 2492483 (13%) 32829.79 12311.47 SOAP/pe_both_k33 2405 20326354 61.483 2835716 (13%) 26706.45 12975.35 SOAP/pe_both_k37 2280 20412482 61.328 1987084 (9%) 28706.97 13993.94 SOAP/pe_both_k39 2271 20809661 61.318 1408958 (6%) 29732.93 14409.50 SOAP/pe_both_k41 2275 20493113 61.295 1777292 (8%) 29447.59 14232.10 velvet/pe_both 3122 19461165 60.702 944011 (4%) 19010.44 9070.62 velvet/single 57772 20825488 60.981 5670 (0%) 1129.67 480.99
What is striking, Velvet gives small number of contigs (and high n50 & n90) with 600-bp library alone! Unfortunately, assembly misses quite a lot of data (18Mb as compared to 20Mb in others).
On the other hand, SOAPdenovo with both libraries gives the best assembly: 2271 contigs, n50 ~30kb and only 6% of Ns.
Do you have any comments on that?
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2 answers
In you stat. info, the situation using both libraries has a double times n50 larger than using only one library, and the contigs number decreased, it seems good performance. Genome assembly , there would be a saturation value of coverage for each libraries, when you try to calculate the n50, whole length and contigs number, the accumulated curve will tell you this.
BTW, as you ask the other assemblers for multiple insert size libraries, I recommend you try these:
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SOAPdenovo (Current version 1.05, 14-02-2011, 2.0 will be released soon which has been announced) - SOAPdenovo is a novel short-read assembly method that can build a de novo draft assembly for the human-sized genomes. The program is specially designed to assemble Illumina GA short reads. It creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost effective way.
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ALLPATH (v2.2, 2-5-2010) - ALLPATHS is the predecessor of ALLPATHS-LG. It works on ~30 base reads.
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Ray (1.6.0, 6-13-2011) - Ray is a parallel software that computes de novo genome assemblies with next-generation sequencing data.-
And I personally prefer SOAPdenovo for illumina short and different insert size PE/MP libraries.--
I am impressed with the improvement you are seeing, because it's not like the 600bp library is going to span a different class of repeats than 300bp library. What is the result of running the 600 alone?
I would continue to explore the Velvet parameter space, as well as turn off pairing to see how much that is being leveraged.
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i am not familiar with 2x45bp - did you hard trim 76bp reads? if so try quality trimming instead using the ucdavis pair-safe trimmer
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SOAP/pe_both_k39 looks best in your result. It uses both libs, and it is the best among the different K-mers you picked for SOAPdenovo. Try different K-mers on velvet as well, using both libs.
I've tried K in range 31-61 with 2 increment, k39 seems to be the best for velvet as well (accordingly to VelvetOptimiser for the best n50 and Lbp as well)