Question: RNA Normalization from DESeq to RPKM
0
gravatar for khadeeja0909
3.6 years ago by
khadeeja090910
Norway
khadeeja090910 wrote:

I have RNA Seq DESeq normalized data.I want to convert it into RPKM.

kindly let me know what should I do?

Any R package etc.

Thank You.

rna-seq next-gen R • 2.3k views
ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by khadeeja090910
1
gravatar for Devon Ryan
3.6 years ago by
Devon Ryan90k
Freiburg, Germany
Devon Ryan90k wrote:

Take the counts, divide them by the gene length in KB (you can probably download this, but if not just google for how to generate it from a GTF file) and then divide by the number of mapped reads in millions.

For what it's worth, edgeR provides an rpkm() function, though once again you'll need to supply the gene lengths.

ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by Devon Ryan90k

Devon, In that case, the normalized read count would be the base mean number generated by the Deseq for each experimental/controle comparision?

ADD REPLYlink modified 3.6 years ago • written 3.6 years ago by tiago2112871.1k

The normalized counts are per-sample.

ADD REPLYlink written 3.6 years ago by Devon Ryan90k

BTW, I should note that if you input normalized counts then you can just divide by a million rather than number of mapped reads in millions. It's best to not adjust for library-size differences twice...

ADD REPLYlink written 3.6 years ago by Devon Ryan90k

If you input DESeq normalized count, then it is not RPKM (Reads Per Kilobase of transcript per Million mapped reads) but something like "Reads Per Kilobase of transcript per Million mapped reads on exons". I'm not saying it is a bad metric, but don't call this RPKM to avoid confusion !

ADD REPLYlink written 3.6 years ago by Carlo Yague4.4k

I hate to break it to you but it's quite likely that most published RPKM values are calculated in this manner. I agree that a different term should probably be used, but that ship has already sailed.

ADD REPLYlink written 3.6 years ago by Devon Ryan90k

While this might be true, I don't think we should encourage use of inaccurate/imprecise terms... this just add to the general confusion with all the FPKM/RPKM/TPM/... things.

ADD REPLYlink written 3.6 years ago by Carlo Yague4.4k
0
gravatar for khadeeja0909
3.6 years ago by
khadeeja090910
Norway
khadeeja090910 wrote:
Total gene read counts were normalized on library size using DESeq method (size factor)
Feature_ID M1_1 M1_2 M2_1 M2_2 M3_1 M3_2 M4_1 M4_2 M5_1 M5_2 M6_1 M6_2 M7_1 M7_2 M8_1 M8_2 M9_1 M9_2 M10_1 M10_2
AT1G01010 82.76 155.63 82.04 120.97 96.56 89.69 148.62 88.95 56.90 112.33 122.04 127.13 119.41 107.63 125.20 105.24 98.49 94.63 55.92 41.66
AT1G01020 287.06 233.44 232.45 326.93 261.60 194.49 478.16 424.96 108.23 241.24 296.39 327.82 405.37 459.73 333.87 300.69 318.63 356.01 539.58 682.54
AT1G01030 20.69 1.35 28.49 26.03 17.96 19.15 10.77 28.33 1.12 7.37 33.62 27.32 10.47 8.21 1.39 8.10 39.59 34.92 50.03 77.97
AT1G01040 1100.40 521.02 961.69 1202.83 898.21 1179.01 1620.81 1694.58 706.82 511.95 1414.69 1671.66 1864.49 1848.93 1295.15 1350.78 1352.74 1394.73 1789.44 1843.60
AT1G01046 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
AT1G01050 1705.56 2801.34 2171.78 1633.89 1871.64 1609.30 1285.88 1171.45 2309.58 2438.19 1463.26 1272.39 1210.87 1132.89 1499.65 1391.25 1760.20 1696.66 1372.49 1527.43

This is my file that I am using Devon. This A.Thaliana data. So simply i divide each counts with transcript length in kb then it will be converted to RPKM?

 

ADD COMMENTlink written 3.6 years ago by khadeeja090910

Divide by a million too, that'll be the M part in RPKM.
 

ADD REPLYlink written 3.6 years ago by Devon Ryan90k

Thank You Devon.

ADD REPLYlink written 3.6 years ago by khadeeja090910
0
gravatar for khadeeja0909
3.6 years ago by
khadeeja090910
Norway
khadeeja090910 wrote:

Thank You so much.

It helped me a lot

ADD COMMENTlink written 3.6 years ago by khadeeja090910
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