De novo Genome Assembly

0. Download your file and gunzip it

Download link: →
The address of my gunzipped file: /nfs/srv/databases/ngs/sophia.veselova/pr14/SRR4240361.fastq

1. Trimmomatic

Terminal commands:

cp *.fa /nfs/srv/databases/ngs/sophia.veselova/pr14/	\| copying all files with the adapters to my directory
cat *.fa > adapters.fasta	\| merging all adapters into one file
java -jar /usr/share/java/trimmomatic.jar SE -phred33 SRR4240361.fastq adcut.fastq ILLUMINACLIP:adapters.fasta:2:7:7	\| removing adapters
java -jar /usr/share/java/trimmomatic.jar SE -phred33 adcut.fastq trimdone.fastq TRAILING:20 MINLEN:30	\| leaving only required length reads without end low quality nucleotides

Output for Trimmomatic:

Input Reads: 7272621 Surviving: 7238064 (99,52%) Dropped: 34557 (0,48%)

Input Reads: 7238064 Surviving: 6881690 (95,08%) Dropped: 356374 (4,92%)

File size:

Name	Size
SRR4240361.fastq	734M
adcut.fastq	730M
trimdone.fastq	690M

2. Velveth

First, I've decided to learn something about velveth, so I ran through this manual and it turned out that velveth is just setting up some data for velvetg to work with.
k-mers - all possible subsequences of length k (in our case it's 29 bc 29 is the closest possible number to 30 and we can't take 30 bc our number must be odd (bc there may be palindromes);
hash length - the length of the k-mers.

Terminal commands:

velveth -help	\| same manual but in terminal
velveth velveth 29 -fastq -short trimdone.fastq	\| velveth output_directory hash_length [[-file_format][-read_type] filename]

P.S. received data is in velveth directory.

3. Velvetg

Then, all data obtained from velveth I used in velvetg. It's the main feature of Velvet where the de Bruijn graph is built then manipulated.

Terminal command:

velvetg velveth

| velvetg output_directory

Output for velvetg:

Final graph has 1222 nodes and n50 of 49972, max 155850, total 690940, using 0/6881690 reads

Files: contigs.fa, stats.txt

N50 = 49972;
Average length = 565
Average coverage = 12

Three longest contigs:

ID	Length	File
3	155850	3.fa
11	85024	11.fa
1	72780	1.fa

Amomalous coverage:

ID	Coverage
73	130,75
121	122,59
472	1,72

4. Analysis

Using megablast I compared 3 longest contigs with Buchnera aphidicola chromosome. Results are below:

NODE_1_length_72780_cov_35.516788

NODE_3_length_155850_cov_33.079514

NODE_11_length_85024_cov_34.670528

Coordinates

Contig	Genome
[57822:65135]	[474667:467412]
[5:2796]	[531590:528794]
[23323:31768]	[508806:500370]
[15452:21628]	[516539:510438]
[2866:8442]	[528679:523105]
[65162:70106]	[467421:462496]
[44408:50485]	[488106:481997]
[51845:57719]	[480660:474844]
[10457:14186]	[521500:517766]
[31884:36161]	[500325:496111]
[37579:38955]	[494864:493487]
[50957:51639]	[481545:480874]
[37327:37445]	[495148:495033]

Coordinates

Contig	Genome
[81937:91416]	[275551:266073]
[110407:121104]	[247596:236918]
[12176:20526]	[341508:333222]
[52503:59856]	[303252:295935]
[73657:81878]	[283706:275566]
[150564:155812]	[207661:202390]
[134400:138502]	[223720:219625]
[41358:45669]	[312179:307878]
[129900:134011]	[228137:224057]
[121202:125731]	[236859:232358]
[125880:129000]	[232057:228944]
[93966:97537]	[263784:260224]
[6511368479]	[291560:288181]
[105728:108930]	[252161:248967]
[37405:40713]	[315982:312679]
[139649:142334]	[218384:215717]
[7060:10399]	[346547:343228]
[30158:34371]	[323043:318826]
[145910:148869]	[212243:209294]
[100315:104637]	[257546:253223]
[23571:26340]	[330003:327227]
[60165:61691]	[295755:294227]
[1672:4246]	[352456:349918]
[26632:28838]	[326950:324747]
[4495:5937]	[349674:348233]
[37:843]	[353822:353014]
[10545:11817]	[343052:341781]
[70269:71603]	[286535:285200]
[22517:23190]	[331006:330333]
[72300:73409]	[285070:283963]
[149315:150205]	[208904:208017]
[138550:139210]	[219491:218821]

Coordinates

Contig	Genome
[62724 :72165]	[398726:389348]
[57496 :62479]	[403823:398904]
[43342 :47936]	[417677:413081]
[14422 :17449]	[445895:442877]
[7198 :11850]	[454069:449411]
[31369 :35447]	[429483:425412]
[55085 :57253]	[406218:404050]
[22353 :25276]	[438139:435267]
[25338 :28767]	[435241:431839]
[49023 :50837]	[412321:410512]
[17579 :19254]	[442817:441135]
[74888 :76355]	[386887:385425]
[39557 :40410]	[421327:420477]
[76419 :77702]	[385420:384182]
[19376 :19557]	[440944:440755]
[19647 :19728]	[440732:440652]

Identity range: from 74.074 to 92.665 %
Evalue range: from 9,88E-163 to 0.0
Strand: +/-

Identity range: from 73,4 to 83,847 %
Evalue range: from 9,88E-163 to 0.0
Strand: +/-

Identity range: from 74,083 to 97,561 %
Evalue range: from 1.92e-165 to 0.0
Strand: +/-

Note:
Assuming data above, the whole lentgth of contig is aligned (reversed). There are also some gaps.

Note:
Assuming data above, the whole length of contig is aligned (reversed). There are also some gaps.

Note:
Assuming data above, almost whole length of contig (except 1-7K region, see hit table) is aligned (reversed). There are also some gaps.

Download hit table

Other data

ID	Total Score	Max score	E-value	Query cover	Ident
1	31952	4047	0.00	78%	77%
3	63791	6154	0.00	75%	79%
11	26639	3605	0.00	54%	74%

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