AspSeq assembly progress

Raw data

Illumina Paired End:

This was our initial dataset. Sequencing was done on a HiSeq using older chemistry (several years ago). This data has some GC bias issues, due to the chemistry.

Library Name	Coverage	MeanRL	MedianRL	Count	MeanQ	MedianQ	MeanGC	MedianGC	Insert Size
PE150	11.44	101	101	54275444	31.77	36	0.4079	0.4046	150
PE150.1	11.44	101	101	54275444	32.58	36.99	0.4073	0.4043	150
PE150-1	26.41	101	101	125234671	32.4	36.58	0.3859	0.3861	150
PE150-1.1	26.41	101	101	125234671	30.58	35.96	0.3864	0.3861	150
PE300	21.42	101	101	101587753	28.92	35	0.3769	0.3762	300
PE300.1	21.42	101	101	101587753	30.78	36	0.3769	0.3762	300
PE650	13.4	101	101	63533295	28.3	35.01	0.3791	0.382	650
PE650.1	13.4	101	101	63533295	30.84	36.02	0.3783	0.3792	650

454 reads

For the initial project, we also generated about 10X 454 data.

Library Name	Coverage	MeanRL	MedianRL	Count	MeanQ	MedianQ	MeanGC	MedianGC	Insert Size
asp201Run1se_1	0.2848	287.5	288.1	474412	31.08	34.85	0.3607	0.3641	NA
asp201Run1se_2	0.2932	303.3	306.4	463040	31.44	35.24	0.3617	0.3651	NA
asp201Run2pe_1	0.2496	292.1	313.5	409339	28.94	30.8	0.3786	0.3695	3000
asp201Run2pe_2	0.2557	298.1	321	410843	29.09	31	0.3795	0.3699	3000
asp201Run3se_1	0.000635	130.8	77.35	2326	23.8	23.93	0.4796	0.4809	NA
asp201Run3se_2	0.0001951	134.8	81.94	693	26.01	25.24	0.4788	0.4833	NA
asp201Run4se_1	0.4005	346.6	404.7	553438	31.07	34.37	0.4061	0.3855	NA
asp201Run4se_2	0.3629	337	390.4	515844	30.53	33.15	0.4113	0.3894	NA
asp201Run5se_1	0.5258	342	372.6	736322	29.61	32.07	0.3464	0.3494	NA
asp201Run5se_2	0.5788	342.5	376.1	809422	30.02	32.65	0.3504	0.3529	NA
asp201Run6se_1	0.3801	355.2	390.7	512577	30.39	33.66	0.3441	0.3474	NA
asp201Run6se_2	0.4044	364.2	402.2	531805	30.64	34.06	0.3444	0.3473	NA
asp201Run7se_1	0.3812	346	385.1	527682	29.98	32.57	0.3487	0.3521	NA
asp201Run7se_2	0.3919	348.8	388	538231	29.9	32.4	0.3494	0.3526	NA
asp201Run8se_1	0.3791	307.3	329.5	590877	28.68	30.66	0.349	0.3519	NA
asp201Run8se_2	0.44	306.5	328.9	687637	28.53	30.35	0.3482	0.3512	NA
GWDW1HR01	0.7442	533.4	583.6	668353	27.6	28.59	0.3646	0.3641	NA
GWDW1HR02	0.7451	541.6	595.3	658997	27.71	28.77	0.3641	0.3639	NA
GWLD3LU01	0.7276	524.9	568.8	663912	28.57	30.15	0.3643	0.3644	NA
GWLD3LU02	0.6257	483.7	521	619620	27.83	28.78	0.3667	0.3663	NA
GWLD5AX01	0.365	447.2	489.3	390891	27.44	28.06	0.3703	0.3699	NA
GWLD5AX02	0.5504	498.6	541.1	528744	28.3	29.59	0.3676	0.3674	NA
GWLFG7T01	0.7119	557.3	639.7	611862	28.65	30.44	0.3634	0.3644	NA
GWLFG7T02	0.4897	511.9	582	458205	28.66	30.29	0.3662	0.367	NA

Illumina experimental runs

As part of our collaboration with the Swedish central sequencing facility, they tested new protocols on their HiSeq and MiSeq machines to sequence long overlapping fragments. The fragments here are 450bp in total, 300bp overlapping. The HiSeq machine was run in Rapid Mode.

Library Name	Coverage	MeanRL	MedianRL	Count	MeanQ	MedianQ	MeanGC	MedianGC	"Insert" size
MiSeq-300	18.06	301	301	28740790	33.24	37	0.3499	0.3505	-150
MiSeq-300.1	18.06	301	301	28740790	28.6	36.16	0.3553	0.3525	-150
HiSeq-300	113.1	301	301	1.8e+08	34.05	38	0.3464	0.3488	-150
HiSeq-300.1	113.1	301	301	1.8e+08	28.75	38	0.3491	0.3484	-150
HiSeq-300.2	113.6	301	301	180772624	34.19	38	0.3461	0.3488	-150
HiSeq-300.3	113.6	301	301	180772624	29.01	38	0.3484	0.3455	-150

Jumping reads

To scaffold the initial assemblies, we generated some Mate Pair libraries. With exception to the 10Kb library, these suffer from high PE contamination and overall mediocre quality:

Library Name	Coverage	MeanRL	MedianRL	Count	MeanQ	MedianQ	MeanGC	MedianGC	Insert size
3KbMP	22.15	101	101	1.05e+08	32.66	36.74	0.3632	0.3586	3000
3KbMP.1	22.15	101	101	1.05e+08	32.48	37	0.3635	0.3632	3000
10KbMP	2.811	49	49	27481587	36.76	38.96	0.3653	0.3673	10000
10KbMP.1	2.811	49	49	27481587	32.89	37.02	0.3761	0.3784	10000
5KbMP	2.959	101	101	14031890	34.45	36.4	0.3643	0.3564	5000
5KbMP.1	2.959	101	101	14031890	33.64	36.22	0.3641	0.3564	5000
3KbMP.2	8.774	101	101	41611057	32.31	35.28	0.367	0.3663	3000
3KbMP.3	8.774	101	101	41611057	34.9	38.04	0.3664	0.3663	3000
5KbMP.2	12.01	81.87	93.95	70277953	37.6	39	0.3485	0.3536	5000
5KbMP.3	12.02	81.89	93.96	70277953	37.34	38.96	0.3484	0.3532	5000

Fosmid pools

Recently, we ran a pilot to sequence fosmid pools and fosmid ends, which included ~8x PacBio (filtered subreads) of 5x1000 fosmids as well as fosmid end sequencing, generating jumping libraries with 40Kb insert sizes. Only about 5-10% of the initial fosmid end data maps with expected insert sizes.

Library Name	Coverage	MeanRL	MedianRL	Count	MeanQ	MedianQ	MeanGC	MedianGC	Insert Size
pb_162-1	1.568	5729	5861	131109	43.2	44.59	0.3922	0.3769	NA
pb_162-2	1.726	7931	7964	104220	43.09	44	0.3991	0.3832	NA
pb_162-3	1.668	7749	7742	103112	43.07	44	0.3977	0.3823	NA
pb_162-4	1.64	7518	7466	104519	43.04	44	0.3994	0.3826	NA
pb_162-5	1.302	6992	6707	89210	42.98	44	0.3983	0.3826	NA
FE1	4.918	151	151	15602157	35.96	38	0.4081	0.4159	40000
FE1.1	4.918	151	151	15602157	35.18	38	0.4082	0.4106	40000
FE2	4.968	151	151	15758362	35.95	38	0.4082	0.4106	40000
FE2.1	4.968	151	151	15758362	35.29	38	0.408	0.4106	40000

Genomic PacBio

In addition to the previous PacBio data, we have generated 60X PacBio (filtered subreads)

Library Name	Coverage	MeanRL	MedianRL	Count	MeanQ	MedianQ	MeanGC	MedianGC
pb_158	60.28	8062	7771	3581635	42.6	43.29	0.3648	0.3539

Assembly statistics

The assembly that is currently in use (Potra v1.1) is a hybrid assembly utilizing the initial Illumina PE data, the 454 data, as well as the Illumina Mate Pair data. Two newer generations of the assembly are listed here. The first is an assembly of the high coverage overlapping Illumina data using DISCOVAR de novo, the second is a PacBio only FALCON assembly. The expected genome size is 479Mbp, as estimated by flow cytometry.

Assembly	# Scaffolds	N50	Total length
Potra v1.1	204318	44Kbp (Scaffold)	387Mbp
DISCOVAR	44375	31Kbp (Contig)	491Mbp
FALCON	4845	484Kbp (Contig)	477Mbp

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