Tool for PacBio Denovo assembly of diploid plant genome (size 500mb) having around 52x coverage

Question: Tool for PacBio Denovo assembly of diploid plant genome (size 500mb) having around 52x coverage

4.2 years ago by

India

Hello Everyone

I have query regarding tool for de novo assembly of PacBio data. I have plant genome data at 52X coverage. The genome size is around 500 mb

I used HGAP (RS_HGAP_assembly2, RS_HGAP_assembly3 , RS_preassembly 2) tool through SMRT portal it giving me error of "ERROR! Reading fasta files greater than 4Gbytes is not supported" . It is not supporting large gnome size

Then I used falcon it run successfully but in assembly folder the all files are empty except preads.ovl, 2-asm-falcon/run_falcon_asm.sh.log

Can you please suggest me tool for assembly for data having 52x coverage and predicted genome size is 500mb .

Thank you

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ADD COMMENT • link •

modified 4.2 years ago by lexnederbragt • 1.2k • written 4.2 years ago by Sudhir Jadhao • 60

4.2 years ago by

rhall • 160

United States

rhall • 160 wrote:

Falcon is probably the best option, rather then try other assemblers I would try to diagnose what went wrong with the Falcon run, it's likely just a parameter problem.

What is the output of DBstats ./1-preads_ovl/preads.db

With 52x of PacBio data, hybrid assembly with something like DBG2OLC would not add anything.

ADD COMMENT • link written 4.2 years ago by rhall • 160

Thank you for kind reply,

I rerun falcon on whole data . it run successfully . but the output is in kb

preads.db log

files = 106
3749 out.00001 prolog
7455 out.00002 prolog
12044 out.00003 prolog
18311 out.00004 prolog
24536 out.00005 prolog
30657 out.00006 prolog
36772 out.00007 prolog
42550 out.00008 prolog
46543 out.00009 prolog
50505 out.00010 prolog
55975 out.00011 prolog
63720 out.00012 prolog
71217 out.00013 prolog
75404 out.00014 prolog
79358 out.00015 prolog
83326 out.00016 prolog
87104 out.00017 prolog
90888 out.00018 prolog
94756 out.00019 prolog
98699 out.00020 prolog
102804 out.00021 prolog
106537 out.00022 prolog
110015 out.00023 prolog
113750 out.00024 prolog
117753 out.00025 prolog
122021 out.00026 prolog
126277 out.00027 prolog
129746 out.00028 prolog
132839 out.00029 prolog
136150 out.00030 prolog
140462 out.00031 prolog
144600 out.00032 prolog
148781 out.00033 prolog
152037 out.00034 prolog
155257 out.00035 prolog
158580 out.00036 prolog
162379 out.00037 prolog
166088 out.00038 prolog
169529 out.00039 prolog
172859 out.00040 prolog
176340 out.00041 prolog
180329 out.00042 prolog
184182 out.00043 prolog
187650 out.00044 prolog
191011 out.00045 prolog
194399 out.00046 prolog
197757 out.00047 prolog
201247 out.00048 prolog
204729 out.00049 prolog
208079 out.00050 prolog
211316 out.00051 prolog
214869 out.00052 prolog
218730 out.00053 prolog
222538 out.00054 prolog
226689 out.00055 prolog
230930 out.00056 prolog
235133 out.00057 prolog
239246 out.00058 prolog
243418 out.00059 prolog
248062 out.00060 prolog
252833 out.00061 prolog
257530 out.00062 prolog
262077 out.00063 prolog
266686 out.00064 prolog
271244 out.00065 prolog
275749 out.00066 prolog
280553 out.00067 prolog
285516 out.00068 prolog
290487 out.00069 prolog
295389 out.00070 prolog
300290 out.00071 prolog
304943 out.00072 prolog
309542 out.00073 prolog
314121 out.00074 prolog
319207 out.00075 prolog
324500 out.00076 prolog
328657 out.00077 prolog
332975 out.00078 prolog
337067 out.00079 prolog
341947 out.00080 prolog
346923 out.00081 prolog
351301 out.00082 prolog
355614 out.00083 prolog
360051 out.00084 prolog
364202 out.00085 prolog
368858 out.00086 prolog
373229 out.00087 prolog
377188 out.00088 prolog
381736 out.00089 prolog
386442 out.00090 prolog
390553 out.00091 prolog
395058 out.00092 prolog
399367 out.00093 prolog
403795 out.00094 prolog
408248 out.00095 prolog
412911 out.00096 prolog
417611 out.00097 prolog
422363 out.00098 prolog
427270 out.00099 prolog
432073 out.00100 prolog
436938 out.00101 prolog
441737 out.00102 prolog
446443 out.00103 prolog
451264 out.00104 prolog
456158 out.00105 prolog
458034 out.00106 prolog
blocks = 13
size = 200 cutoff = 500 all = 0
0 0
29554 29554
59672 59672
89818 89818
128917 128917
166272 166272
202887 202887
241204 241204
281728 281728
322513 322513
363304 363304
404378 404378
444177 444177
458034 458034

Config file

[General]
# list of files of the initial bas.h5 files
input_fofn = input.fofn
#input_fofn = preads.fofn

input_type = raw
#input_type = preads
job_type= local

# The length cutoff used for seed reads used for initial mapping
length_cutoff = 12000

# The length cutoff used for seed reads usef for pre-assembly
length_cutoff_pr = 12000

jobqueue = your_queue
sge_option_da = -pe smp 8 -q %(jobqueue)s
sge_option_la = -pe smp 2 -q %(jobqueue)s
sge_option_pda = -pe smp 8 -q %(jobqueue)s
sge_option_pla = -pe smp 2 -q %(jobqueue)s
sge_option_fc = -pe smp 24 -q %(jobqueue)s
sge_option_cns = -pe smp 8 -q %(jobqueue)s

pa_concurrent_jobs = 32
ovlp_concurrent_jobs = 32

pa_HPCdaligner_option = -v -dal24 -t16 -e.70 -l1000 -s1000
ovlp_HPCdaligner_option = -v -dal24 -t32 -h60 -e.96 -l500 -s1000

pa_DBsplit_option = -x500 -s200
ovlp_DBsplit_option = -x500 -s200

falcon_sense_option = --output_multi --min_idt 0.70 --min_cov 4 --local_match_count_threshold 2 --max_n_read 200 --n_core 6

overlap_filtering_setting = --max_diff 100 --max_cov 100 --min_cov 20 --bestn 10 --n_core 40

ADD REPLY • link modified 4.2 years ago • written 4.2 years ago by Sudhir Jadhao • 60

How many bases are in the 1-preads_ovl/preads4falcon.fasta file? Two things stand out as being things to change, if the preads4falcon.fasta file does not have >15x of the expected genome size, then the length_cutoff and length_cutoff_pr parameters should be decreased, this will be dependent on your library quality and subread size. The second parameter that needs to be changed is the --min_cov in the overlap_filtering_setting I would set it at 2.

ADD REPLY • link written 4.2 years ago by rhall • 160

Hey thanks I got your point. Now I will rerun process with following parameter just tell me they are good to go.

length_cutoff = 500

length_cutoff_pr = 2500

--min_cov 20

after completing process with above parameter I will get back to you.

And one more thing If you have any good reading material regarding falcon parameter for diploid genome please let me know

Thank you

ADD REPLY • link modified 4.2 years ago • written 4.2 years ago by Sudhir Jadhao • 60

I tried with new parameter Now I got below error

1) raise TaskFailureError("Counted %d failures." %failedJobCount)
TaskFailureError: 'Counted 1 failures.'
No target specified, assuming "assembly" as target

2) File "/home/bionivid-server/.local/lib/python2.7/site-packages/falcon_kit-0.4.0-py2.7-linux-x86_64.egg/falcon_kit/mains/run.py", line 447, in main1
wf.refreshTargets(updateFreq = wait_time) # larger number better for more jobs, need to call to run jobs here or the # of concurrency is changed
File "/usr/local/lib/python2.7/dist-packages/pypeflow-0.1.1-py2.7.egg/pypeflow/controller.py", line 531, in refreshTargets
rtn = self._refreshTargets(task2thread, objs = objs, callback = callback, updateFreq = updateFreq, exitOnFailure = exitOnFailure)
File "/usr/local/lib/python2.7/dist-packages/pypeflow-0.1.1-py2.7.egg/pypeflow/controller.py", line 706, in _refreshTargets
raise TaskFailureError("Counted %d failures." %failedJobCount)
pypeflow.controller.TaskFailureError: 'Counted 1 failures.'

ADD REPLY • link written 4.2 years ago by Sudhir Jadhao • 60

500 and 2500 are too low for the cutoffs, this should be calculated as the sequence length for which ~30x of you expected genome size is covered.

ADD REPLY • link written 4.2 years ago by rhall • 160

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