Methods for quantifying efficiency of nucleic acid extraction and detection
Methods for quantifying the efficiency of nucleic acid extraction from a sample comprising a mixture of species are provided. In some embodiments, the method comprises adding an initial amount of one or more spike-ins to the sample, extracting nucleic acids from the sample, quantifying the amount of the one or more spike-ins in the extracted nucleic acid sample, and comparing the initial amount of the one or more spike-ins to the quantified amount of the one or more spike-ins.
1 . A method for quantifying efficiency of nucleic acid extraction from a soil sample comprising a mixture of species, the method comprising:
providing the soil sample comprising the mixture of species;
adding spike-ins to the soil sample, the spike-ins comprising synthetic nucleic acid sequences, wherein at least one of the synthetic nucleic acid sequences includes a repeating pattern of a plurality of adenine, thymine, cytosine, or guanine content,
wherein the spike-ins comprise an initial amount of a spike-in;
extracting nucleic acids from the soil sample comprising the spike-ins, thereby forming an extracted nucleic acid sample;
obtaining sequencing reads of the extracted nucleic acid sample;
determining a detected amount of each species in the mixture of species in the extracted nucleic acid sample based on the sequencing reads;
quantifying the spike-ins in the extracted nucleic acid sample to generate a quantified amount of the spike-in based on the sequencing reads;
determining a ratio of the initial amount of the spike-in to the quantified amount of the spike-in, thereby quantifying the efficiency of the nucleic acid extraction from the soil sample;
normalizing the detected amount of each species based on the ratio of the initial amount of the spike-in to the quantified amount of the spike-in;
determining that the quantified amount of the spike-in is less than a predetermined threshold amount of the initial amount of the spike-in;
and responsive to determining that the quantified amount of the spike-in is less than the predetermined threshold amount of the initial amount of the spike-in, correlating a characteristic of the soil sample with the efficiency of the nucleic acid extraction.
2 . The method of claim 1 , wherein the soil sample comprising the mixture of species comprises one or more bacterial species, phytoplasma species, viral species, viroid species, rickettsia species, fungal species, helminth species, protozoan, parasite species, and/or pest species.
3 . The method of claim 2 , wherein the one or more bacterial species, phytoplasma species, viral species, viroid species, rickettsia species, fungal species, helminth species, protozoan, parasite species, and/or pest species are plant pathogens.
4 . The method of claim 1 , wherein the synthetic nucleic acid sequences comprise an artificial sequence flanked on each end by a transposon sequence.
5 . The method of claim 1 , wherein the step of adding the spike-ins comprises adding a dilution series of a known species.
6 . The method of claim 1 , wherein the quantifying step comprises detecting a nucleic acid sequence in the spike-ins.
7 . The method of claim 1 , wherein the predetermined threshold amount is 85% or lower.
8 . The method of claim 1 , wherein the predetermined threshold amount is 80% or lower.
9 . The method of claim 1 , wherein the predetermined threshold amount is 70% or lower.
10 . The method of claim 1 , wherein the predetermined threshold amount is 60% or lower.
11 . The method of claim 1 , wherein the predetermined threshold amount is 50% or lower.
12 . A non-transitory computer-readable storage medium storing instructions, the instructions when executed by one or more processors cause the one or more processors to:
quantify spike-ins in an extracted nucleic acid sample to generate a quantified amount of a spike-in of the spike-ins,
wherein the extracted nucleic acid sample is formed by extracting nucleic acids from a soil sample comprising a mixture of species added with the spike-ins,
wherein the spike-ins comprise an initial amount of the spike-in,
wherein the spike-ins comprise synthetic nucleic acid sequences, wherein at least one of the synthetic nucleic acid sequences includes a repeating pattern of a plurality of adenine, thymine, cytosine, or guanine content;
determining a detected amount of each species in the mixture of species in the extracted nucleic acid sample based on sequencing reads of the extracted nucleic acid sample;
determining a ratio of the initial amount of the spike-in to the quantified amount of the spike-in, thereby quantifying efficiency of nucleic acid extraction from the soil sample;
normalizing the detected amount of each species based on the ratio of the initial amount of the spike-in to the quantified amount of the spike-in;
determining that the quantified amount of the spike-in is less than a predetermined threshold amount of the initial amount of the spike-in;
and responsive to determining that the quantified amount of the spike-in is less than the predetermined threshold amount of the initial amount of the spike-in, correlating a characteristic of the soil sample with the efficiency of the nucleic acid extraction.
13 . The non-transitory computer-readable storage medium of claim 12 , wherein the soil sample comprising the mixture of species comprises one or more bacterial species, phytoplasma species, viral species, viroid species, rickettsia species, fungal species, helminth species, protozoan, parasite species, and/or pest species.
14 . The non-transitory computer-readable storage medium of claim 12 , wherein the predetermined threshold amount is 85% or lower.