Testing RNAseq parameters

Aligner	GRCh38-local	GRCh38	T2T
star_salmon	completed successfully	completed successfully	completed successfully
star_rsem	did not complete successfully	completed successfully	completed successfully
hisat2	did not complete successfully	did not complete successfully	not tested

SALMON vs. RSEM gene counts:

No significant changes in feature count values observed for “gene_counts” produced by RSEM or SALMON (see figure below). Comparison based on 45,315 commonly reported genes by SALMON (n=51,912) and RSEM (n=45,706).
Most significant changes observed are similar across all samples, hence, differential expression analysis should produce similar results for SALMON and RSEM.

Table: Stats of uniquely reported gene counts by a SALMON or RSEM.

Tool

Unique genes

No expression

(Zero counts)

Difference above (average count >=50)

Difference above (average count >=100)

Difference above (average count >=500)

SALMON

6,597

3,277 (49.7%)

140 (2.1%)

82 (1.2%)

30 (0.4%)

RSEM

391

216 (55.2%)

29 (7.4%)

23 (5.9%)

9 (2.3%)

Example - RSEM unique genes:

gene_id transcript_id(s)        SRR20622172     SRR20622173     SRR20622174     SRR20622175     SRR20622176     SRR20622177     SRR20622178     SRR20622179     SRR20622180
ENSMUSG00000003178      ENSMUST00000077159      978.00  889.00  712.00  716.00  911.00  819.00  1065.00 1214.00 1566.00
ENSMUSG00000029333      ENSMUST00000031276,ENSMUST00000166484,ENSMUST00000166632,ENSMUST00000168092     386.00  256.00  173.00  160.00  358.00  212.00  419.00  508.00  553.00
ENSMUSG00000032134      ENSMUST00000034653      12.00   20.00   374.00  445.00  16.00   539.00  29.00   64.00   98.00
ENSMUSG00000035349      ENSMUST00000021384      204.00  177.00  103.00  112.00  187.00  137.00  43.00   56.00   67.00
ENSMUSG00000038194      ENSMUST00000127256,ENSMUST00000137350,ENSMUST00000155591        7.00    16.89   15.22   10.81   15.00   11.24   44.72   32.92   40.82
ENSMUSG00000042402      ENSMUST00000045546      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000043858      ENSMUST00000057195,ENSMUST00000107891,ENSMUST00000136861        2727.91 1965.92 668.00  643.00  2571.96 750.93  786.96  1097.95 1152.00
ENSMUSG00000045946      ENSMUST00000056234      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000047490      ENSMUST00000050983      0.00    0.00    4.00    3.00    1.00    5.00    1.00    1.00    0.00
ENSMUSG00000048665      ENSMUST00000054470      0.00    0.00    0.00    1.00    0.00    0.00    0.00    0.00    1.00
ENSMUSG00000050347      ENSMUST00000052897      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000050431      ENSMUST00000051453      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000050604      ENSMUST00000062454      6.00    3.00    3.00    3.00    8.00    4.00    5.00    3.00    6.00
ENSMUSG00000050781      ENSMUST00000062407      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000051052      ENSMUST00000156004      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000052429      ENSMUST00000045325,ENSMUST00000085387,ENSMUST00000107840,ENSMUST00000107842,ENSMUST00000107843,ENSMUST00000125886,ENSMUST00000127531,ENSMUST00000153576 6824.00 4330.00 1250.00 1438.00 6072.00 1652.00 1675.00 2254.00 2492.00
ENSMUSG00000054758      ENSMUST00000067972      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000055099      ENSMUST00000068485      0.00    0.00    1.00    3.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000055694      ENSMUST00000046516      0.00    0.00    0.00    0.00    0.00    0.00    0.00    1.09    0.00
ENSMUSG00000056106      ENSMUST00000070021      4.00    1.00    8.90    3.00    3.00    1.00    5.00    6.00    4.00
ENSMUSG00000056606      ENSMUST00000070814      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000056872      ENSMUST00000077868      0.00    0.00    1.00    1.00    0.00    3.00    0.00    0.00    0.00
ENSMUSG00000057351      ENSMUST00000078761      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000057626      ENSMUST00000076465      2.49    0.00    2.49    2.50    8.69    2.49    3.44    8.99    7.58
ENSMUSG00000057832      ENSMUST00000080925      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000057924      ENSMUST00000072427      56.97   31.00   43.98   51.00   66.00   41.31   60.00   52.00   61.00
ENSMUSG00000059041      ENSMUST00000080251      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000059101      ENSMUST00000079234      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000059227      ENSMUST00000073059      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000059448      ENSMUST00000079465      0.00    0.00    2.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000059511      ENSMUST00000071632      0.00    0.00    2.36    0.00    2.63    0.00    1.91    2.51    0.00
ENSMUSG00000059731      ENSMUST00000071975      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000059996      ENSMUST00000073580      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000060393      ENSMUST00000122096      6.30    1.74    3.69    1.24    3.74    1.19    1.20    4.84    2.58
ENSMUSG00000060559      ENSMUST00000074466      3.00    0.00    16.00   11.00   0.00    14.00   14.00   30.00   30.00
ENSMUSG00000061075      ENSMUST00000077247      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000061305      ENSMUST00000074192      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000061501      ENSMUST00000080031      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00
ENSMUSG00000061777      ENSMUST00000076936      0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00    0.00

Run time comparison

	GRCh38-local star_salmon	GRCh38-local star_rsem	GRCh38 star_salmon	GRCh38 star_rsem	T2T star_salmon	T2T star rsem
Duration	1h 15m 33s	1h 46m 52s	1h 27m 22s	2h 57m 55s	2h 2m 59s	2h 35m 30s
CPU hours	60.1	139.1	54.4	119.3	139.8	163.1
Wall time			01:27:56	02:58:24	02:03:28	02:36:06
Memory			4,194,304 kb	4,194,304 kb	4,194,304 kb	4,194,304 kb